U.S. patent number 10,927,170 [Application Number 15/979,609] was granted by the patent office on 2021-02-23 for anti-claudin 1 monoclonal antibodies for the prevention and treatment of hepatocellular carcinoma.
This patent grant is currently assigned to Institut Hospitalier Universitaire de Strasbourg, Institut National de la Sante et de la Recherche Medicale, Universite de Strasbourg. The grantee listed for this patent is INSERM (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE), INSTITUT HOSPITALIER UNIVERSITAIRE DE STRASBOURG, UNIVERSITE DE STRASBOURG. Invention is credited to Thomas Baumert, Eric Robinet, Mirjam Zeisel.
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United States Patent |
10,927,170 |
Baumert , et al. |
February 23, 2021 |
Anti-claudin 1 monoclonal antibodies for the prevention and
treatment of hepatocellular carcinoma
Abstract
Use of anti-Claudin 1 monoclonal antibodies and pharmaceutical
compositions thereof, for the prevention and/or treatment of
hepatocellular carcinoma in patients suffering from liver disease,
in particular liver disease that is not associated with HCV
infection or in patients who have been cured from HCV infection.
Methods of preventing and/or treating hepatocellular carcinoma by
administration of such a monoclonal antibody, or a pharmaceutical
composition thereof, are also described. Experimental results with
the hepatocarcinoma cell line HuH-7.5.1 are given.
Inventors: |
Baumert; Thomas (Freiburg,
DE), Robinet; Eric (Colmar, FR), Zeisel;
Mirjam (Strasbourg, FR) |
Applicant: |
Name |
City |
State |
Country |
Type |
INSTITUT HOSPITALIER UNIVERSITAIRE DE STRASBOURG
INSERM (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE
MEDICALE)
UNIVERSITE DE STRASBOURG |
Strasbourg
Paris
Strasbourg |
N/A
N/A
N/A |
FR
FR
FR |
|
|
Assignee: |
Universite de Strasbourg
(Strasbourg, FR)
Institut Hospitalier Universitaire de Strasbourg
(Strasbourg, FR)
Institut National de la Sante et de la Recherche Medicale
(Paris, FR)
|
Family
ID: |
1000005376306 |
Appl.
No.: |
15/979,609 |
Filed: |
May 15, 2018 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20180258169 A1 |
Sep 13, 2018 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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15557969 |
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10815298 |
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PCT/EP2016/055942 |
Mar 18, 2016 |
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Foreign Application Priority Data
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Mar 19, 2015 [EP] |
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15159872 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
45/06 (20130101); C07K 16/28 (20130101); A61K
39/39558 (20130101); A61K 39/39558 (20130101); A61K
2300/00 (20130101); C07K 2317/76 (20130101) |
Current International
Class: |
C07K
16/28 (20060101); A61K 39/395 (20060101); A61K
45/06 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 2010/034812 |
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Apr 2010 |
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WO |
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2015/014657 |
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Feb 2015 |
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WO |
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WO 2015/014657 |
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Feb 2015 |
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WO |
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Other References
International Search Report and Written Opinion of the
International Searching Authority dated May 27, 2016, which issued
during prosecution of International Application No.
PCT/EP2016/055942. cited by applicant.
|
Primary Examiner: Goddard; Laura B
Attorney, Agent or Firm: Morgan, Lewis & Bockius LLP
Parent Case Text
RELATED PATENT APPLICATIONS
The present application is a Continuation of U.S. application Ser.
No. 15/557,969 filed Sep. 13, 2017, which was filed pursuant to 35
U.S.C. .sctn. 371 as a U.S. National Phase Application of
International Patent Application No. PCT/EP2016/055942, which was
filed on Mar. 18, 2016, claiming the benefit of priority to
European Patent Application number EP 15 159 872.9, which was filed
on Mar. 19, 2015. The entire contents of each of the aforementioned
patent applications is incorporated herein by reference in its
entirety.
Claims
What is claimed is:
1. A method of treating a non-alcoholic fatty liver disease (NAFLD)
in a subject, comprising a step of administering to the subject in
need thereof, an effective amount of an anti-Claudin 1 antibody, or
a biologically active fragment thereof, wherein the anti-Claudin 1
antibody is a monoclonal anti-Claudin 1 antibody secreted by a
hybridoma cell line deposited at the Deutsche Sammlung von
Mikroorganismen and Zellkulturen (DSMZ) on Jul. 29, 2008 under an
Accession Number selected from the group consisting of DSM ACC2931,
DSM ACC2932, DSM ACC2933, DSM ACC2934, DSM ACC2935, DSM ACC2936,
DSM ACC2937, and DSM ACC2938, or wherein the anti-Claudin 1
antibody, or the biologically active fragment thereof, comprises
the six complementary determining regions (CDRs) of a monoclonal
antibody secreted by one of said hybridoma cell lines.
2. The method according to claim 1, wherein the NAFLD is
non-alcoholic steatohepatitis (NASH).
3. The method according to claim 1, wherein the NAFLD is
non-HCV-associated.
4. The method according to claim 3, wherein the subject has never
been infected with HCV or has been cured from HCV infection.
5. The method according to claim 1, wherein the progression of the
NAFLD is slowed down, reduced, stopped, alleviated, or
reversed.
6. The method according to claim 1, wherein the anti-Claudin 1
antibody is a monoclonal antibody.
7. The method according to claim 1, wherein the anti-Claudin 1
antibody, or biologically active fragment thereof, is humanized,
de-immunized or chimeric.
8. The method according to claim 1, wherein the anti-Claudin 1
antibody, or biologically active fragment thereof, is in the form
of a pharmaceutical composition comprising an effective amount of
the anti-Claudin 1 antibody, or biologically active fragment
thereof, and at least one pharmaceutically acceptable
excipient.
9. The method according to claim 8, wherein the pharmaceutical
composition further comprises an additional therapeutic agent.
10. The method according to claim 9, wherein the additional
therapeutic agent is selected from the group consisting of
anti-viral agents, anti-inflammatory agents, immunomodulatory
agents, analgesics, antimicrobial agents, kinase inhibitors,
molecules interfering with signalling, antibacterial agents,
antibiotics, antioxidants, antiseptic agents, anti-cancer agents
and combinations thereof.
Description
BACKGROUND OF THE INVENTION
Hepatocellular carcinoma (HCC) is the second leading and fastest
rising cause of cancer death worldwide (International Agency for
Research on Cancer; GLOBOCAN 2012: Estimated Cancer Incidence,
Mortality and Prevalence Worldwide in 2012--webpage:
globocan.iarc.fr). HCC accounts for more than 500,000 new cases per
year and nearly as many deaths due to poor disease prognosis.
Chronic hepatitis C virus (HCV) infection is the most important
risk factor for developing liver cirrhosis and HCC (El-Serag, N
Engl J Med., 2011, 365(12): 1118-1127). It is estimated that
approximately 3% of the world population is chronically infected
with HCV (World Health Organization). Other major risk factors for
HCC include infection with hepatitis B virus (HBV), alcoholic liver
disease, and non-alcoholic fatty liver disease. Less common causes
include hereditary hemochromatosis, alpha 1-antitrypsin deficiency,
auto-immune hepatitis, some porphyrias, Wilson's disease, aflatoxin
exposure. The distribution of these risk factors among patients
with HCC is highly variable, depending on geographic region, and on
race or ethnic group. Most of these risk factors lead to the
formation and progression of cirrhosis, which is present in 80 to
90% of patients with HCC. The 5-year cumulative risk for the
development of HCC in patients with cirrhosis ranges between 5% and
30%, depending on the cause, region or ethnic group, and stage of
cirrhosis. In 2011, end-stage liver disease and HCC resulted in
6,342 liver transplants associated with costs of more than 1
billion US dollars for the procedure alone (see NIH webpage:
optn.transplant.hrsa.gov/latestData/step2.asp).
Although HCC may be avoided by addressing the underlying cause in
the early stage of the disease, strategies to prevent HCC in
patients with established cirrhosis and advanced fibrosis, in which
the risk of HCC persists despite treatment of the underlying cause,
are lacking. Indeed, even curing HCV infection does not eliminate
the risk of HCC development when advanced fibrosis is already
present (van der Meer et al., JAMA, 2012, 308(24): 2584-2593).
Currently, curative treatment options for patients with cirrhotic
HCC are mainly limited to liver transplantation, an impractical,
invasive and resource-intensive solution. Given the extremely
frequent tumor recurrence after surgical treatment and absence of
efficient medical treatment strategies, prevention of HCC
development in patients with advanced liver fibrosis is considered
to be the most effective strategy to substantially impact on
patient survival (Hoshida et al., J Hepatol., 2014, 61(1S):
S79-S90; Hoshida et al., Curr Cancer Drug Targets, 2012,
12(9):1129-1159).
In light of the increasing economic burden of patients with
cirrhosis and associated HCC, novel strategies to prevent and treat
HCC in patients with advanced liver disease are therefore urgently
needed.
SUMMARY OF THE INVENTION
The present invention relates to systems and strategies for the
prevention and/or treatment of hepatocellular carcinoma (HCC)
irrespective of the etiology. In particular the present invention
is directed to the use of anti-Claudin-1 antibodies for preventing
and/or treating hepatocellular carcinoma, including hepatocellular
carcinoma that is not associated with HCV infection and
hepatocellular carcinoma that has developed, or that is susceptible
of developing, after HCV infection has been cured. Analyzing
virus-induced cell signalling and a 186-liver gene signature which
predicts HCC risk in cirrhotic patients of various etiologies, the
present Applicants have shown that an anti-Claudin 1 monoclonal
antibody, which they had previously developed and shown to cure
chronic HCV infection without detectable adverse effects (EP 08 305
597 and WO 2010/034812), interferes with liver cell signalling and
reverses a patient-derived HCC risk signature in a liver cell-based
model system. Modulation of signalling and transcriptional
reprogramming was found to be independent of the antiviral activity
of the antibody, indicating that the anti-Claudin 1 monoclonal
antibody acts directly onto oncogenic pathways. Indeed, performing
mechanistic studies, the Applicants have demonstrated that the
antibody impairs the EGFR-MAPK signalling pathway and expression of
inflammatory response genes, which have been suggested as drivers
for hepatocarcinogenesis. Compared to antiviral agents and other
candidate compounds for HCC chemoprotection, the anti-Claudin 1
monoclonal antibody was the most potent to reverse the HCC
high-risk signature.
Consequently, in one aspect, the present invention provides an
anti-Claudin 1 antibody, or a biologically active fragment thereof,
for use in the prevention or treatment of a non-HCV-associated
hepatocellular carcinoma in a subject, i.e., in a subject that has
never been infected with HCV or in a subject that has been cured
from HCV infection.
In certain embodiments, the non-HCV associated hepatocellular
carcinoma is associated with hepatitis B virus (HBV) infection,
alcoholism, non-alcoholic fatty liver disease (NAFLD), hereditary
hemochromatosis, alpha 1 antitrypsin deficiency, porphyria cutanea
tarda, Wilson's disease, tyrosinemia, glycogen storage diseases,
autoimmune hepatitis, primary biliary cirrhosis, or exposure to
aflatoxins. In other embodiments, the non-HCV-associated
hepatocellular carcinoma is hepatocellular carcinoma of unknown
origin.
In certain embodiments, the anti-Claudin 1 antibody is a polyclonal
antibody. In other embodiments, the anti-Claudin 1 antibody is a
monoclonal antibody.
In certain embodiments, the anti-Claudin 1 antibody is a monoclonal
antibody secreted by a hybridoma cell line co-deposited by INSERM
and GENOVAC at the DSMZ on Jul. 29, 2008 under an Accession Number
selected from the group consisting of DSM ACC2931, DSM ACC2932, DSM
ACC2933, DSM ACC2934, DSM ACC2935, DSM ACC2936, DSM ACC2937, and
DSM ACC2938. In other embodiments, the anti-Claudin 1 antibody
comprises the six complementary determining regions (CDRs) of a
monoclonal antibody secreted by a hybridoma cell line co-deposited
by INSERM and GENOVAC at the DSMZ on Jul. 29, 2008 under an
Accession Number selected from the group consisting of DSM ACC2931,
DSM ACC2932, DSM ACC2933, DSM ACC2934, DSM ACC2935, DSM ACC2936,
DSM ACC2937, and DSM ACC2938.
In certain embodiments, the anti-Claudin 1 antibody is humanized,
de-immunized or chimeric.
A biologically active fragment of an anti-Claudin 1 antibody is a
fragment that retains the biological property of the antibody to
interfere with liver cell signalling and to reverse a
patient-derived HCC risk signature.
More generally, the present invention encompasses the use of any
molecule that comprises an anti-Claudin-1 antibody, or a
biologically active fragment thereof, including chimeric
antibodies, humanized antibodies, de-immunized antibodies and
antibody-derived molecules comprising at least one complementary
determining region (CDR) from either a heavy chain or light chain
variable region of an anti-Claudin-1 monoclonal antibody secreted
by a hybridoma cell line, including molecules such as Fab
fragments, F(ab').sub.2 fragments, Fd fragments, Sc antibodies
(single chain antibodies), diabodies, individual antibody light
single chains, individual antibody heavy chains, chimeric fusions
between antibody chains and other molecules, and antibody
conjugates, such as antibodies conjugated to a diagnostic agent
(detectable moiety) or therapeutic agent, so long as these
antibody-related molecules retain the biological property to
interfere with liver cell signalling and/or to reverse a
patient-derived HCC risk signature and/or to prevent or treat
non-HCV-associated hepatocellular carcinoma.
In a related aspect, the present invention provides a method for
preventing hepatocellular carcinoma in a subject suffering from a
non-HCV-associated liver disease, said method comprising a step of
administering to the subject in need thereof an effective amount of
an anti-Claudin 1 antibody or a biologically active fragment
thereof. As indicated above, the subject suffering from liver
disease has never been infected with HCV or has been cured from HCV
infection.
In certain embodiments, the underlying cause of the non-HCV
associated liver disease is selected from the group consisting of
hepatitis B virus (HBV) infection, alcoholism, non-alcoholic fatty
liver disease (NAFLD), hereditary hemochromatosis, alpha 1
antitrypsin deficiency, porphyria cutanea tarda, Wilson's disease,
tyrosinemia, glycogen storage diseases, autoimmune hepatitis,
primary biliary cirrhosis, and exposure to aflatoxins. In other
embodiments, the non-HCV-associated liver disease is of unknown
origin.
In another related aspect, the present invention provides a method
of treating non-HCV-associated hepatocellular carcinoma in a
subject, said method comprising a step of administering to the
subject in need thereof an effective amount of an anti-Claudin 1
antibody or a biologically active fragment thereof. As indicated
above, the subject suffering from hepatocellular carcinoma has
never been infected with HCV or has been cured from HCV
infection.
In certain embodiments, the non-HCV-associated hepatocellular
carcinoma carcinoma is associated with hepatitis B virus (HVB)
infection, alcoholism, non-alcoholic fatty liver disease (NAFLD),
hereditary hemochromatosis, alpha 1 antitrypsin deficiency,
porphyria cutanea tarda, Wilson's disease, tyrosinemia, glycogen
storage diseases, autoimmune hepatitis, primary biliary cirrhosis,
or exposure to aflatoxins. In other embodiments, the
non-HCV-associated hepatocellular carcinoma is hepatocellular
carcinoma of unknown origin.
The anti-Claudin 1 antibodies and biologically active fragments
thereof that may be used in the practice of the method of
prevention of the present invention and in the method of treatment
of the present invention are as described above.
In another aspect, the present invention provides a pharmaceutical
composition comprising an effective amount of an anti-Claudin 1
antibody, or a biologically active fragment thereof, and at least
one pharmaceutically acceptable carrier or excipient, for use in
the prevention or treatment of a non-HCV associated hepatocellular
carcinoma in a subject, i.e., a subject that has never been
infected with HCV or in as subject that has been cured from HCV
infection.
In certain embodiments, the non-HCV associated hepatocellular
carcinoma is associated with hepatitis B virus infection,
alcoholism, non-alcoholic fatty liver disease (NAFLD), hereditary
hemochromatosis, alpha 1 antitrypsin deficiency, porphyria cutanea
tarda, Wilson's disease, tyrosinemia, glycogen storage diseases,
autoimmune hepatitis, primary biliary cirrhosis, or exposure to
aflatoxins. In other embodiments, the non-HCV-associated
hepatocellular carcinoma is hepatocellular carcinoma of unknown
origin.
The anti-Claudin 1 antibodies and biologically active fragments
thereof that may be present in a pharmaceutical composition
according to the present invention are as described above.
In certain embodiments, a pharmaceutical composition according to
present invention further comprises an additional therapeutic
agent. The additional therapeutic agent may be is selected from the
group consisting of anti-viral agents, anti-inflammatory agents,
immunomodulatory agents, analgesics, antimicrobial agents,
antibacterial agents, antibiotics, antioxidants, antiseptic agents,
anti-cancer agents and combinations thereof.
In a related aspect, the present invention provides an anti-Claudin
1 antibody, or a biologically active fragment thereof, for the
manufacture of a medicament for the prevention and/or treatment of
non-HCV-associated hepatocellular carcinoma in a subject.
These and other objects, advantages and features of the present
invention will become apparent to those of ordinary skill in the
art having read the following detailed description of the preferred
embodiments.
BRIEF DESCRIPTION OF THE DRAWING
FIGS. 1A-C. Persistent HCV infection-induced HCC 186-gene signature
is reversed following CLDN1-specific mAb treatment in
Huh7.5.1.sup.dif cells. A. Huh7.5.1 cells were differentiated into
hepatocyte-like Huh7.5.1.sup.dif cells, persistently infected using
HCV Jc1 and subjected to erlotinib or CLDN1-specific mAb treatment
or no treatment on day 7 post-infection, then subjected to
transcriptomics analysis. B. Immunodetection of HCV E2 protein (day
7 of infection). Dapi of nuclei staining in blue. Scale bar 50
.mu.m. C. GSEA showing the reversal of the HCC high- and low-risk
genes following erlotinib and CLDN1-specific mAb treatment.
Compared to erlotinib, CLDN1 mAb showed better efficacy in
reversing the HCC high risk profile.
FIGS. 2A-B. CLDN1-specific mAb reverses HCC high-risk genes more
potently than direct-acting antivirals or other HCC chemoprevention
candidate compounds independently of the viral load. A.
Huh7.5.1.sup.dif cells were HCV Jc1 infected. On day 7
post-infection, cells were treated with different compounds. Total
cellular RNA was isolated and subjected to NanoString analysis.
Treatment of HCV Jc1-infected Huh7.5.1.sup.dif cells with DAA (1 nM
DCV+1 .mu.M SOF), interferon alpha-2a (10 IU/ml), erlotinib (0.1
.mu.M), and pioglitazone (1 .mu.M), partially reverses the HCC
high-risk genes as shown by GSEA. Control treatment with metformin
(Met, 3 .mu.M) had no effect. Treatment with CLDN1-specific mAb (1,
10 and 100 .mu.g/ml) potently reverses HCC-high risk genes.
Heatmaps show the significance of HCC high-/low-risk gene signature
induction or suppression. B. Reversal of the gene signature by
CLDN1-specific mAb is independent of viral load. Relative HCV RNA
expressions (normalized to GAPDH) were analyzed. HCV load in
cell-based model, mean.+-.SD, n=3. FC--Fold change,
DCV--Daclatasvir, SOF--Sofosbuvir.
FIGS. 3A-G. CLDN1-specific mAb impairs EGFR/MAPK signaling and
expression in human liver cells. A, B. Huh7.5.1 cells were infected
with HCV Jc1 and harvested for proteomic analyses. Infection with
HCV Jc1-E2.sup.FLAG results in increased EGFR phosphorylation. A.
Receptor tyrosine kinase (RTK) phosphorylation was assessed in cell
lysates using the Human Phospho-RTK Array Kit (R&D Systems). B.
Quantification of dot blot intensities of phosphorylated proteins
using Image J software. Mean.+-.SEM of integrated dot blot
densities, n=3. C-E. EGFR and EGF mRNA expression (relative to
GAPDH mRNA) in uninfected (Ctrl) and HCV Jc1-infected
Huh7.5.1.sup.dif cells (C; n=9); uninfected (Ctrl) and HBV-infected
HepG2-NTCP cells (D; n=12); Huh7.5.1.sup.dif cells incubated in
absence (Ctrl) or presence of 40 mM ethanol (E; n=6). Mean
percentage.+-.SEM is shown. * Mann-Whitney U-test
(p-value<0.01). F. CLDN1-specific mAb impairs HCV-induced host
cell signaling. Detection of kinase phosphorylation in chronically
HCV Jc1-infected Huh7.5.1 cells treated with control or
CLDN1-specific mAbs (100 .mu.g/mL; 24 h) using human phosphokinase
arrays. p-Erk1/2 highlighted by black squares in F was quantified
using Image J software (NIH). Results are shown as mean.+-.SEM of
integrated dot blot densities from 2 independent experiments
performed in duplicate. G. Reversal of EGFR-MAPK signal pathway
expression by CLDN1-specific mAb treatment. Huh7.5.1.sup.dif cells
were HCV Jc1 infected. Total RNA was isolated and subjected to
NanoString analysis. Plots represent GSEA enrichment scores of EGFR
signaling in cancer retrieved from oncogenic signatures database
(EGFR_UP.V1_UP).
FIGS. 4A-B. EGFR and MAPK signaling pathways are suppressed
following CLDN1-specific mAb treatment of HCV Jc1-infected
Huh7.5.1.sup.dif cells. In two independent experiments,
Huh7.5.1.sup.dif cells were HCV Jc1 infected and treated with mAbs
as described above. Total cellular RNA was isolated and subjected
to NanoString analysis. Differentially expressed genes were
selected. Intensity expression values were normalized and log
transformed; differentially expressed genes have FDR
p-values<0.05 and fold change of .+-.1.9. Ingenuity Pathway
Analysis (IPA.RTM., webpage: qiagen.com/ingenuity) was used for the
generation of network analysis. Differentially expressed genes
belonging to A. EGFR and B. MAPK-signaling pathways are suppressed
following CLDN1-specific mAb treatment. Filled nodes with genes in
bold represent differentially expressed genes belonging to EGFR or
MAPK signaling pathway, rounded rectangle meaning up-regulated and
circles meaning down-regulated following CLDN1-specific mAb
treatment. Squares represent predicted targets; genes in bold and
black outline belong to EGFR or MAPK signaling pathway. Solid and
dashed lines indicate direct and indirect interactions,
respectively.
FIG. 5. CLDN1-specific mAb treatment modulates NF-.kappa.B
inflammatory signaling pathway in HCV Jc1-infected Huh7.5.1.sup.dif
cells. In two independent experiments, Huh7.5.1.sup.dif cells were
HCV Jc1 infected and treated with mAbs as described above. Total
cellular RNA was isolated and subjected to NanoString analysis.
Differentially expressed genes were selected. The networks were
generated through the use of Ingenuity Pathway Analysis (IPA.RTM.,
webpage: qiagen.com/ingenuity). NF-.kappa.B signaling pathway is
modulated following CLDN1-specific mAb treatment. Filled nodes with
genes in bold represent differentially expressed genes belonging to
NF-.kappa.B signaling pathway, rounded rectangle meaning
up-regulated and circles meaning down-regulated following
CLDN1-specific mAb treatment. Squares represent predicted targets;
genes in bold and black outline belong to NF-.kappa.B signaling
pathway. Solid and dashed lines indicate direct and indirect
interactions, respectively.
FIG. 6. Treatment of HCV Jc1-infected Huh7.5.1.sup.dif cells with
CLDN1-specific mAb suppresses liver disease-induced genes. In two
independent experiments, Huh7.5.1.sup.dif cells were HCV Jc1
infected and treated with mAbs as described above. Total cellular
RNA was isolated and subjected to NanoString analysis.
Differentially expressed genes were selected. Ingenuity Pathway
Analysis (IPA.RTM., webpage: qiagen.com/ingenuity) was used for the
generation of network analysis. Liver disease-induced genes
extracted by IPA software based on Ingenuity Knowledge Base are
shown to be suppressed following CLDN1-specific mAb treatment.
Nodes represent differentially expressed genes, rounded rectangle
meaning up-regulated and circles meaning down-regulated following
CLDN1-specific mAb treatment. Squares represent predicted targets.
Solid and dashed lines indicate direct and indirect interactions,
respectively. Genes with black outline are involved in liver
diseases curated in IPA Knowledge Base.
FIGS. 7A-C. Patient-derived panetiology 32-gene HCC risk signature
is reversed following CLDN1-specific mAb treatment in HBV-infected
HepG2-NTCP cells. A. HepG2-NTCP cells were infected with
serum-derived HBV and treated with human CLDN1-specific mAb or
control Ab for 7 days. B. HBV infection was confirmed through
quantification of relative HBV pregenomic (pg) RNA expression by
qRT-PCR (mean.+-.SD; n=3). C. Heatmap showing the
suppression/induction of expression of HCC high- and low-risk
genes, respectively, following human CLDN1-specific mAb treatment.
Expression of the HCC-risk signature was assessed using Biomark HD,
high-throughput RT-PCR technology. In scale bar, white indicates
enrichment of suppression, black indicates enrichment of
induction.
FIGS. 8A-B. Patient-derived panetiology 32-gene HCC risk signature
is reversed following CLDN1-specific mAb treatment in
ethanol-treated liver cells. A. Huh7.5.1 cells were differentiated
into hepatocyte-like Huh7.5.1.sup.dif cells, exposed chronically to
ethanol (40 mM) and treated with human CLDN1-specific mAb or
control Ab for the 10 days of exposure. B. Heatmap showing the
suppression/induction of expression of HCC high- and low-risk
genes, respectively, following human CLDN1-specific mAb treatment.
HCC-risk signature was assessed using Biomark HD, high-throughput
RT-PCR technology. In scale bar, white indicates enrichment of
suppression, black indicates enrichment of induction.
FIGS. 9A-C. A Warburg-like metabolic shift associated with
increased cancer risk is reversed following human CLDN1-specific
mAb treatment in HCV-infected Huh7.5.1.sup.dif cells. A. Analysis
of polar metabolites was performed in Huh7.5.1.sup.dif cells
persistently infected with HCV. Ten days after HCV infection,
metabolites were extracted and further analyzed by mass
spectrometry. B. Liver cell lactate flux. Negative values:
accumulation outside the cells. C. Heatmap and hierarchical
clustering showing top 15 detected metabolites.
FIGS. 10A-C. CLDN1-specific mAb treatment reverses epithelial to
mesenchymal (EMT) transition regulators in virus-infected liver
cells. A. Huh7.5.1.sup.dif cells were persistently infected using
HCV Jc1 and treated with CLDN1-specific mAb or control Ab treatment
for 3 days following 7 days of infection. B. Relative expression of
Snail1 (SNAI1), Snail2 (SNAI2), and ZEB1 (ZEB1) upon CLDN1-specific
mAb or control mAb treatment. C. Heatmap showing expression of
genes involved in EMT. CLDN1-specific mAb treatment reversed the
gene expression pattern typically observed in EMT. The genes shown
belong to the 186-gene HCC-risk signature. In scale bar, white
indicates enrichment of suppression, black indicates enrichment of
induction. Results represent one experiment performed in
triplicate. ES: Enrichment Score.
FIGS. 11A-E. Prevention and treatment of liver disease by CLDN1
specific mAb in a DEN mouse model for liver disease and HCC. A.
Approach used. C3H/He mice (n=10) received a single injection of
DEN (D on the graph). Eighteen weeks post DEN injection and before
treatment with antibody, two mice were sacrificed for baseline
analyses (). From week 18 until week 23, the remaining 8 mice were
subjected to treatment with mouse CLDN1-specific Ab mIgG3 (Y) (n=4;
treatment group) or not treated (n=4; control group). One week
after the last antibody treatment, livers were harvested for
post-treatment analyses (). Liver tissue was fixed and stained with
either hematoxillin/eosin (B, C, E) or Masson's trichrome (D). B.
Liver disease at baseline prior to antibody treatment. Eighteen
weeks post DEN injection and before treatment with antibody, two
mice were sacrificed. Livers of mice were harvested, fixed and
stained with hematoxillin/eosin. Arrows show focal areas of
steatosis in the liver of all mice. Magnification .times.200. C, D
and E. Liver disease post treatment with CLDN1-specific mAb. C.
Livers were harvested post treatment at week 23 and stained with
hematoxillin/eosin. Arrows show areas of steatosis exclusively in
control mice but not in anti-CLDN1 treated mice. Magnification
.times.200. D. Masson's trichrome staining of the livers of two out
of four mice per group, confirming the presence of steatosis
(arrows) in control mice while steatosis is not or barely
detectable in mice treated with CLDN1-specific mAb. Two
magnifications (.times.50 and .times.200) are shown. E.
Hematoxillin/eosin staining of a liver tumor in a mouse of the
control group at two different magnifications (.times.50 and
.times.200). No tumors were detected in CLDN1-specific antibody
treated mice. Images are representative of the entire liver.
Identification numbers of the mice are indicated on each slide
(D-E). Size bars: 200 .mu.m for .times.50 magnification and 50
.mu.m for .times.200 magnification.
DEFINITIONS
Throughout the specification, several terms are employed that are
defined in the following paragraphs.
As used herein, the term "subject" refers to a human or another
mammal (e.g., primate, dog, cat, goat, horse, pig, mouse, rat,
rabbit, and the like), that can develop hepatocellular carcinoma,
but may or may not be suffering from the disease. Non-human
subjects may be transgenic or otherwise modified animals. In many
embodiments of the present invention, the subject is a human being.
In such embodiments, the subject is often referred to as an
"individual" or a "patient" The term "individual" does not denote a
particular age, and thus encompasses newborns, children, teenagers,
and adults. The term "patient" more specifically refers to an
individual suffering from a disease. In the practice of the present
invention, a patient will generally be diagnosed with a liver
disease.
The term "treatment" is used herein to characterize a method or
process that is aimed at (1) delaying or preventing the onset of a
disease or condition (e.g., hepatocellular carcinoma); (2) slowing
down or stopping the progression, aggravation, or deterioration of
the symptoms of the disease or condition (e.g., liver disease); (3)
bringing about amelioration of the symptoms of the disease or
condition; or (4) curing the disease or condition. A treatment may
be administered prior to the onset of the disease or condition, for
a prophylactic or preventive action. Alternatively or additionally,
a treatment may be administered after initiation of the disease or
condition, for a therapeutic action.
The terms "hepatocellular carcinoma" and "HCC" are used herein
interchangeably. They refer to the most common type of liver
cancer, also called malignant hepatoma. As used herein, the terms
"HCV-associated hepatocellular carcinoma" and HCV-associated liver
disease" refers to hepatocellular carcinoma and liver disease
respectively that are secondary to infection with hepatitis C virus
(HCV). As used herein, the term "non-HCV-associated hepatocellular
carcinoma" refers to hepatocellular carcinoma that develops, or
that is susceptible of developing, in a patient who has never been
infected with HCV. "Non-HCV-associated hepatocellular carcinoma"
also includes hepatocellular carcinoma that develops, or that is
susceptible of developing, in a patient who has been cured from HCV
infection. Similarly, the term "non-HCV-associated liver disease"
refers to a liver disease that has developed in a patient who has
never been infected with HCV or in patient who has been cured from
HCV infection. Examples of non-HCV-associated hepatocellular
carcinoma/liver disease include hepatocellular carcinoma/liver
disease secondary to hepatitis B virus (HBV) infection, alcoholic
liver disease, non-alcoholic fatty liver disease, hereditary
hemochromatosis, alpha 1-antitrypsin deficiency, auto-immune
hepatitis, some porphyrias, Wilson's disease, aflatoxin exposure,
type 2 diabetes, obesity, etc. . . . , as well as hepatocellular
carcinoma/liver disease of unknown origin.
A "pharmaceutical composition" is defined herein as comprising an
effective amount of at least one anti-Claudin 1 antibody (or a
biologically active fragment thereof), and at least one
pharmaceutically acceptable carrier or excipient.
As used herein, the term "effective amount" refers to any amount of
a compound, agent, antibody, or composition that is sufficient to
fulfil its intended purpose(s), e.g., a desired biological or
medicinal response in a cell, tissue, system or subject. For
example, in certain embodiments of the present invention, the
purpose(s) may be: to prevent the onset of hepatocellular
carcinoma, to slow down, alleviate or stop the progression,
aggravation or deterioration of the symptoms of liver disease or
hepatocellular carcinoma; to bring about amelioration of the
symptoms of the disease, or to cure the hepatocellular
carcinoma.
The term "pharmaceutically acceptable carrier or excipient" refers
to a carrier medium which does not interfere with the effectiveness
of the biological activity of the active ingredient(s) and which is
not excessively toxic to the host at the concentration at which it
is administered. The term includes solvents, dispersion, media,
coatings, antibacterial and antifungal agents, isotonic agents, and
adsorption delaying agents, and the like. The use of such media and
agents for pharmaceutically active substances is well known in the
art (see for example "Remington's Pharmaceutical Sciences", E. W.
Martin, 18.sup.th Ed., 1990, Mack Publishing Co.: Easton, Pa.,
which is incorporated herein by reference in its entirety).
The term "human Claudin-1 or human CLDN1" refers to a protein
having the sequence shown in NCBI Accession Number NP_066924, or
any naturally occurring variants commonly found in HCV permissive
human populations. The term "extracellular domain" or "ectodomain"
of Claudin-1 refers to the region of the Claudin-1 sequence that
extends into the extracellular space (i.e., the space outside a
cell).
The term "antibody", as used herein, refers to any immunoglobulin
(i.e., an intact immunoglobulin molecule, an active portion of an
immunoglobulin molecule, etc.) that binds to a specific epitope.
The term encompasses monoclonal antibodies and polyclonal
antibodies. All derivatives and fragments thereof, which maintain
specific binding ability, are also included in the term. The term
also covers any protein having a binding domain, which is
homologous or largely homologous to an immunoglobulin-binding
domain. These proteins may be derived from natural sources, or
partly or wholly synthetically produced.
The term "specific binding", when used in reference to an antibody,
refers to an antibody binding to a predetermined antigen.
Typically, the antibody binds with an affinity of at least
1.times.10.sup.7 M.sup.-1, and binds to the predetermined antigen
with an affinity that is at least two-fold greater than the
affinity for binding to a non-specific antigen (e.g., BSA,
casein).
The term "isolated", as used herein in reference to a protein or
polypeptide, means a protein or polypeptide, which by virtue of its
origin or manipulation is separated from at least some of the
components with which it is naturally associated or with which it
is associated when initially obtained. By "isolated", it is
alternatively or additionally meant that the protein or polypeptide
of interest is produced or synthesized by the hand of man.
The terms "protein", "polypeptide", and "peptide" are used herein
interchangeably, and refer to amino acid sequences of a variety of
lengths, either in their neutral (uncharged) forms or as salts, and
either unmodified or modified by glycosylation, side-chain
oxidation, or phosphorylation. In certain embodiments, the amino
acid sequence is a full-length native protein. In other
embodiments, the amino acid sequence is a smaller fragment of the
full-length protein. In still other embodiments, the amino acid
sequence is modified by additional substituents attached to the
amino acid side chains, such as glycosyl units, lipids, or
inorganic ions such as phosphates, as well as modifications
relating to chemical conversions of the chains such as oxidation of
sulfydryl groups. Thus, the term "protein" (or its equivalent
terms) is intended to include the amino acid sequence of the
full-length native protein, or a fragment thereof, subject to those
modifications that do not significantly change its specific
properties. In particular, the term "protein" encompasses protein
isoforms, i.e., variants that are encoded by the same gene, but
that differ in their pI or MW, or both. Such isoforms can differ in
their amino acid sequence (e.g., as a result of allelic variation,
alternative splicing or limited proteolysis), or in the
alternative, may arise from differential post-translational
modification (e.g., glycosylation, acylation, phosphorylation).
The term "analog", as used herein in reference to a protein, refers
to a polypeptide that possesses a similar or identical function as
the protein but need not necessarily comprise an amino acid
sequence that is similar or identical to the amino acid sequence of
the protein or a structure that is similar or identical to that of
the protein. Preferably, in the context of the present invention, a
protein analog has an amino acid sequence that is at least 30%,
more preferably, at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95% or 99% identical to the amino acid sequence
of the protein.
The term "fragment" or the term "portion", as used herein in
reference to a protein, refers to a polypeptide comprising an amino
acid sequence of at least 5 consecutive amino acid residues
(preferably, at least about: 10, 15, 20, 25, 30, 35, 40, 50, 60,
70, 80, 90, 100, 125, 150, 175, 200, 250 or more amino acid
residues) of the amino acid sequence of a protein. The fragment of
a protein may or may not possess a functional activity of the
protein.
The term "biologically active", as used herein to characterize a
protein variant, analog or fragment, refers to a molecule that
shares sufficient amino acid sequence identity or homology with the
protein to exhibit similar or identical properties to the protein.
For, example, in many embodiments of the present invention, a
biologically active fragment of an anti-Claudin 1 antibody is a
fragment that retains the ability of the antibody to interfere with
liver cell signalling and to reverse a patient-derived HCC risk
signature.
The term "homologous" (or "homology"), as used herein, is
synonymous with the term "identity" and refers to the sequence
similarity between two polypeptide molecules or between two nucleic
acid molecules. When a position in both compared sequences is
occupied by the same base or same amino acid residue, the
respective molecules are then homologous at that position. The
percentage of homology between two sequences corresponds to the
number of matching or homologous positions shared by the two
sequences divided by the number of positions compared and
multiplied by 100. Generally, a comparison is made when two
sequences are aligned to give maximum homology. Homologous amino
acid sequences share identical or similar amino acid sequences.
Similar residues are conservative substitutions for, or "allowed
point mutations" of, corresponding amino acid residues in a
reference sequence. "Conservative substitutions" of a residue in a
reference sequence are substitutions that are physically or
functionally similar to the corresponding reference residue, e.g.
that have a similar size, shape, electric charge, chemical
properties, including the ability to form covalent or hydrogen
bonds, or the like. Particularly preferred conservative
substitutions are those fulfilling the criteria defined for an
"accepted point mutation" as described by Dayhoff et al. ("Atlas of
Protein Sequence and Structure", 1978, Nat. Biomed. Res.
Foundation, Washington, D.C., Suppl. 3, 22: 354-352).
The terms "labeled", "labeled with a detectable agent" and "labeled
with a detectable moiety" are used herein interchangeably. These
terms are used to specify that an entity (e.g., an antibody) can be
visualized, for example, following binding to another entity (e.g.,
an antigen). Preferably, a detectable agent or moiety is selected
such that it generates a signal which can be measured and whose
intensity is related to the amount of bound entity. Methods for
labeling proteins and polypeptides, including antibodies, are
well-known in the art. Labeled polypeptides can be prepared by
incorporation of or conjugation to a label, that is directly or
indirectly detectable by spectroscopic, photochemical, biochemical,
immunochemical, electrical, optical or chemical means, or any other
suitable means. Suitable detectable agents include, but are not
limited to, various ligands, radionuclides, fluorescent dyes,
chemiluminescent agents, microparticles, enzymes, colorimetric
labels, magnetic labels, and haptens.
The terms "approximately" and "about", as used herein in reference
to a number, generally include numbers that fall within a range of
10% in either direction of the number (greater than or less than
the number) unless otherwise stated or otherwise evident from the
context (except where such number would exceed 100% of a possible
value).
Detailed Description of Certain Preferred Embodiments
As mentioned above, the present invention concerns the use of
anti-claudin 1 antibodies for the prevention and/or treatment of
hepatocellular carcinoma, in particular for the prevention and/or
treatment of hepatocellular carcinoma that is not
HCV-associated.
I--Anti-Claudin-1 Antibodies
The present Applicants have previously developed monoclonal
antibodies directed against human Claudin-1 and demonstrated that
these monoclonal antibodies cure HCV infection in vivo without
detectable adverse effects (EP 08 305 597 and WO 2010/034812). They
have now demonstrated that these monoclonal antibodies interfere
with liver cell signaling and reverse a patient-derived HCC risk
signature in a liver cell-based model system, and that the
modulation of signaling and transcriptional reprogramming is
independent of the antiviral activity of the antibody.
Human Claudin 1 (CLDN1) is a tight junction protein expressed in
various tissues. In hepatocytes, it plays an important role in
forming barrier separating blood and bile (Zona et al., Viruses,
2014, 6(2): 875-892). CLDN1 has been shown to play a dual role in
liver disease that is HCV-associated: it is an essential host
factor for HCV infection serving as a viral cell entry factor
required for initiation, dissemination and maintenance of infection
(Evans et al., Nature, 2007, 446(7137): 801-805; Mailly et al.,
"Clearance of persistent hepatitis C virus infection using a
claudin-1-targeting monoclonal antibody", Nat Biotech, 2015, in
iress). Moreover, CLDN1 has been reported to be involved in
carcinogenesis via modulation of cell signaling (Suh et al.,
Oncogene, 2013, 32(41): 4873-4882) or via induction of the
expression of matrix metalloproteinases (Oku et al., Cancer Res,
2006, 66(10): 5251-5257). Furthermore, CLDN1 expression has been
shown to be increased in HCC compared to non-diseased liver tissue
(Stebbing et al., Oncogene, 2013, 32(41): 4871-4872).
Anti-Claudin 1 antibodies that can be used in the practice of the
present invention include any antibody which was raised against
Claudin 1 and which can be shown to interfere with liver cell
signaling and to reverse a patient-derived HCC risk signature, for
example in a liver cell-based model system.
Examples of anti-Claudin 1 antibodies that can be used in the
practice of the present invention include, in particular, the
polyclonal and monoclonal anti-CLDN1 antibodies that were developed
by the present Applicants (see EP 08 305 597 and WO 2010/034812,
Fofana et al., Gastroenterology, 2010, 139(3): 953-64, 964.e1-4).
As described in these documents, eight monoclonal antibodies have
been produced by genetic immunization and shown to efficiently
inhibit HCV infection by targeting the extracellular domain of
Claudin-1. Using an infectious HCV model system and primary human
hepatocytes, these monoclonal anti-CLDN1 antibodies have been
demonstrated to efficiently inhibit HCV infection of all major
genotypes as well as highly variable HCV quasispecies in individual
patients. Furthermore, these antibodies efficiently blocked entry
of highly infectious HCV escape variants that were resistant to
neutralizing antibodies in six patients with HCV re-infection
during liver transplantation. The monoclonal anti-Claudin 1
antibodies are called OM-4A4-D4, OM-7C8-A8, OM-6D9-A6, OM-7D4-C1,
OM-6E1-B5, OM-3E5-B6, OM-8A9-A3, and OM-7D3-B3. Thus, suitable
anti-Claudin 1 antibodies are monoclonal antibodies secreted by any
one of the hybridoma cell lines deposited by INSERM (one of the
Applicants) and GENOVAC at the DSMZ (Deutsche Sammlung von
Mikro-organismen und Zellkuturen GmbH, Inhoffenstra e 7 B, 38124
Braunschweig, Germany) on Jul. 29, 2008 under Accession Numbers DSM
ACC2931, DSM ACC2932, DSM ACC2933, DSM ACC2934, DSM ACC2935, DSM
ACC2936, DSM ACC2937, and DSM ACC2938 (described in EP 08 305 597
and WO 2010/034812).
Other examples of suitable anti-Claudin 1 antibodies include those
disclosed in European Pat. No. EP 1 167 389, U.S. Pat. No.
6,627,439, in international patent application published under No.
WO 201/132307 and in international patent applications published
under No. WO 2015/014659 and No. WO 2015/014357, and in Yamashita
et al., J. Pharmacol. Exp. Ther., 2015, 353(1): 112-118.
The anti-Claudin 1 antibodies suitable for use in the present
invention may be polyclonal antibodies or monoclonal
antibodies.
Instead of using the hybridomas described above as a source of the
antibodies, the anti-Claudin 1 antibodies may be prepared by any
other suitable method known in the art. For example, an
anti-Claudin 1 monoclonal antibody may be prepared by recombinant
DNA methods. These methods generally involve isolation of the genes
encoding the desired antibody, transfer of the genes into a
suitable vector, and bulk expression in a cell culture system. The
genes or DNA encoding the desired monoclonal antibody may be
readily isolated and sequenced using conventional procedures (e.g.,
using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). Hybridoma cell lines may serve as a preferred source
of such DNA. Suitable host cells for recombinant production of
antibodies include, but are not limited to, appropriate mammalian
host cells, such as CHO, HeLa, or CV1. Suitable expression plasmids
include, without limitation, pcDNA3.1 Zeo, pIND(SP1), pREP8 (all
commercially available from Invitrogen, Carlsbad, Calif., USA), and
the like. The antibody genes may be expressed via viral or
retroviral vectors, including MLV-based vectors, vaccinia
virus-based vectors, and the like. Cells may be grown using
standard methods, in suitable culture media such as, for example,
DMEM and RPMI-1640 medium. The anti-Claudin 1 antibodies may be
expressed as single chain antibodies. Isolation and purification of
recombinantly produced antibodies may be performed by standard
methods. For example, an anti-Claudin 1 monoclonal antibody may be
recovered and purified from cell cultures by protein A
purification, ammonium sulphate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, such as Protein A column,
hydroxylapatite chromatography, lectin chromatography, or any
suitable combination of these methods. High performance liquid
chromatography (HPLC) can also be employed for purification.
Alternatively, an anti-Claudin 1 antibody for use according to the
present invention may be obtained from commercial sources.
In certain embodiments, an anti-Claudin 1 antibody is used in its
native form. In other embodiments, it is truncated (e.g., via
enzymatic cleavage or other suitable method) to provide
immunoglobulin fragments or portions, in particular, fragments or
portions that are biologically active. Biologically active
fragments or portions of an anti-Claudin 1 antibody include
fragments or portions that retain the ability of the antibody to
interfere with liver cell signaling and reverse a patient-derived
HCC risk signature, for example in a liver cell-based model system
such as the 186-liver gene signature system used by the present
Applicants (see Examples below), and/or the ability to prevent
hepatocellular carcinoma and/or treat hepatocellular carcinoma.
A biologically active fragment or portion of an anti-Claudin 1
antibody may be a Fab fragment or portion, a F(ab').sub.2 fragment
or portion, a variable domain, or one or more CDRs (complementary
determining regions) of the antibody (for example an antibody that
comprises all 6 CDRs of an anti-Claudin 1 monoclonal antibody.
Alternatively, a biologically active fragment or portion of an
anti-Claudin 1 antibody may be derived from the carboxyl portion or
terminus of the antibody protein and may comprise an Fc fragment,
an Fd fragment or an Fv fragment.
Anti-Claudin 1 antibody fragments of the present invention may be
produced by any suitable method known in the art including, but not
limited to, enzymatic cleavage (e.g., proteolytic digestion of
intact antibodies) or by synthetic or recombinant techniques.
F(ab').sub.2, Fab, Fv and ScFv (single chain Fv) antibody fragments
can, for example, be expressed in and secreted from mammalian host
cells or from E. coli. Antibodies can also be produced in a variety
of truncated forms using antibody genes in which one or more stop
codons have been introduced upstream of the natural stop site. The
various portions of antibodies can be joined together chemically by
conventional techniques, or can be prepared as a contiguous protein
using genetic engineering techniques.
Anti-Claudin 1 antibodies (or biologically active fragments
thereof) suitable for use according to the present invention may be
produced in a modified form, such as a fusion protein (i.e., an
immunoglobulin molecule or portion linked to a polypeptide entity).
Preferably, the fusion protein retains the biological property of
the antibody. A polypeptide entity to be fused to an anti-Claudin 1
antibody, or a biologically active fragment thereof, may be
selected to confer any of a number of advantageous properties to
the resulting fusion protein. For example, the polypeptide entity
may be selected to provide increased expression of the recombinant
fusion protein. Alternatively or additionally, the polypeptide
entity may facilitate purification of the fusion protein, for
example, by acting as a ligand in affinity purification. A
proteolytic cleavage site may be added to the recombinant protein
so that the desired sequence can ultimately be separated from the
polypeptide entity after purification. The polypeptide entity may
also be selected to confer an improved stability to the fusion
protein, when stability is a goal. Examples of suitable polypeptide
entities include, for example, polyhistidine tags, that allow for
the easy purification of the resulting fusion protein on a nickel
chelating column. Glutathione-S-transferase (GST), maltose B
binding protein, or protein A are other examples of suitable
polypeptide entities.
An anti-Claudin 1 antibody for use according to the present
invention may be re-engineered so as to optimize stability,
solubility, in vivo half-life, or ability to bind additional
targets. Genetic engineering approaches as well as chemical
modifications to accomplish any or all of these changes in
properties are well known in the art. For example, the addition,
removal, and/or modification of the constant regions of an antibody
are known to play a particularly important role in the
bioavailability, distribution, and half-life of therapeutically
administered antibodies. The antibody class and subclass,
determined by the Fc or constant region of the antibody (which
mediates effector functions), when present, imparts important
additional properties.
Additional fusion proteins of the invention may be generated
through the techniques of DNA shuffling well known in the art (see,
for example, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721;
5,834,252; and 5,837,458).
Anti-Claudin 1 antibodies suitable for use according to the present
invention may also be "humanized": sequence differences between
rodent antibodies and human sequences can be minimized by replacing
residues which differ from those in the human sequences by
site-directed mutagenesis of individual residues or by grafting of
entire regions or by chemical synthesis. Humanized antibodies can
also be produced using recombinant methods. In the humanized form
of the antibody, some, most or all of the amino acids outside the
CDR regions are replaced with amino acids from human immunoglobulin
molecules, while some, most or all amino acids within one or more
CDR regions are unchanged. Small additions, deletions, insertions,
substitutions or modifications of amino acids are permissible as
long as they do not significantly modify the biological activity of
the resulting antibody. Suitable human "replacement" immunoglobulin
molecules include IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgA, IgM,
IgD or IgE molecules, and fragments thereof. Alternatively, the
T-cell epitopes present in rodent antibodies can be modified by
mutation (de-immunization) to generate non-immunogenic rodent
antibodies that can be applied for therapeutic purposes in humans
(see webpage: accurobio.com).
Anti-Claudin 1 antibodies (or biologically active variants or
fragments thereof) suitable for use according to the present
invention may be functionally linked (e.g., by chemical coupling,
genetic fusion, non-covalent association or otherwise) to one or
more other molecular entities. Methods for the preparation of such
modified antibodies (or conjugated antibodies) are known in the art
(see, for example, "Affinity Techniques. Enzyme Purification: Part
B", Methods in Enzymol., 1974, Vol. 34, Jakoby and Wilneck (Eds.),
Academic Press: New York, N.Y.; and Wilchek and Bayer, Anal.
Biochem., 1988, 171: 1-32). Preferably, molecular entities are
attached at positions on the antibody molecule that do not
interfere with the binding properties of the resulting conjugate,
e.g., positions that do not participate in the specific binding of
the antibody to its target.
The antibody molecule and molecular entity may be covalently,
directly linked to each other. Or, alternatively, the antibody
molecule and molecular entity may be covalently linked to each
other through a linker group. This can be accomplished by using any
of a wide variety of stable bifunctional agents well known in the
art, including homofunctional and heterofunctional linkers.
In certain embodiments, an anti-Claudin 1 antibody (or a
biologically active fragment thereof) for use according to the
present invention is conjugated to a therapeutic moiety. Any of a
wide variety of therapeutic moieties may be suitable for use in the
practice of the present invention including, without limitation,
cytotoxins (e.g., cytostatic or cytocidal agents), therapeutic
agents, and radioactive metal ions (e.g., alpha-emitters and
alpha-emitters attached to macrocyclic chelators such as DOTA).
Cytotoxins or cytotoxc agents include any agent that is detrimental
to cells. Examples include, but are not limited to, paclitaxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, thymidine kinase,
endonuclease, RNAse, and puromycin and fragments, variants or
homologs thereof. Therapeutic agents include, but are not limited
to, antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin and doxorubicin), antibiotics (e.g.,
dactinomycin, bleomycin, mithramycin, and anthramycin), and
anti-mitotic agents (e.g., vincristine and vinblastine).
Other therapeutic moieties include proteins or polypeptides
possessing a desired biological activity. Such proteins include,
but are not limited to, toxins (e.g., abrin, ricin A, alpha toxin,
pseudomonas exotoxin, diphtheria toxin, saporin, momordin, gelonin,
pokeweed antiviral protein, alpha-sarcin and cholera toxin);
proteins such as tumor necrosis factor, alpha-interferon,
beta-interferon, nerve growth factor, platelet derived growth
factor, tissue plasminogen activator; apoptotic agents (e.g.,
TNF-.alpha., TNF-.beta.) or, biological response modifiers (e.g.,
lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2),
interleukin-6 (IL-6), granulocyte macrophage colony stimulating
factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), or
other growth factors).
Alternatively or additionally, an antibody of the present invention
(or a biologically active fragment thereof) may be conjugated to a
detectable agent. Any of a wide variety of detectable agents can be
used in the practice of the present invention, including, without
limitation, various ligands, radionuclides (e.g. .sup.3H,
.sup.125I, .sup.131I, and the like), fluorescent dyes (e.g.,
fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin,
allophycocyanin, o-phthalaldehyde and fluorescamine),
chemiluminescent agents (e.g., luciferin, luciferase and aequorin),
microparticles (such as, for example, quantum dots, nanocrystals,
phosphors and the like), enzymes (such as, for example, those used
in an ELISA, i.e., horseradish peroxidase, beta-galactosidase,
luciferase, alkaline phosphatase), colorimetric labels, magnetic
labels, and biotin, dioxigenin or other haptens and proteins for
which antisera or monoclonal antibodies are available.
Other molecular entities that can be conjugated to an antibody of
the present invention (or a biologically active fragment thereof)
include, but are not limited to, linear or branched hydrophilic
polymeric groups, fatty acid groups, or fatty ester groups.
Thus, in the practice of the present invention, anti-Claudin 1
antibodies can be used under the form of full length antibodies,
biologically active variants or fragments thereof, chimeric
antibodies, humanized antibodies, and antibody-derived molecules
comprising at least one complementary determining region (CDR) from
either a heavy chain or light chain variable region of an
anti-Claudin 1 antibody, including molecules such as Fab fragments,
F(ab').sub.2 fragments, Fd fragments, Fabc fragments, Sc antibodies
(single chain antibodies), diabodies, individual antibody light
single chains, individual antibody heavy chains, chimeric fusions
between antibody chains and other molecules, and antibody
conjugates, such as antibodies conjugated to a therapeutic agent or
a detectable agent. Preferably, anti-Claudin 1 antibody-related
molecules according to the present invention will be shown to
interfere with liver cell signaling and to reverse a
patient-derived HCC risk signature (such as the 186-liver gene
signature system used by the present Applicants--see Examples).
One skilled in the art will understand that other compounds
targeting Claudin-1, can be used in the practice of the present
invention, including, but not limited to, small molecules and
siRNAs.
II--Treatment or Prevention of Hepatocellular Carcinoma
A. Indications
The present Applicants have shown that anti-Claudin 1 antibodies
are more potent than antiviral agents and other candidate compounds
for HCC chemoprotection at reversing HCC high-risk signature.
Therefore, anti-Claudin 1 antibodies, or biologically active
fragments thereof, may be used in prophylactic and therapeutic
methods to prevent and/or treat hepatocellular carcinoma.
Methods of treatment of the present invention may be accomplished
using an anti-Claudin 1 antibody, or a biologically active fragment
thereof, or a pharmaceutical composition comprising such an
antibody or fragment (see below). These methods generally comprise
administration of an effective amount of an anti-Claudin-1
antibody, or biologically active fragment thereof, or of a
pharmaceutical composition thereof, to a subject in need thereof.
Administration may be performed using any of the administration
methods known to one skilled in the art (see below).
In particular, the present invention provides a method for
preventing a patient suffering from a liver disease from developing
hepatocellular carcinoma. The liver disease or pathology may be
inflammation of the liver, liver fibrosis, and/or cirrhosis.
In the practice of the present invention, the underlying cause of
the liver disease is not HCV infection. Thus, the invention
provides a method for preventing and/or treating non-HCV-associated
hepatocellular carcinoma, i.e., for preventing and/or treating
hepatocellular carcinoma that develops, or that is susceptible of
developing, in a patient who has never been infected with HCV, or
in a patient who has been cured from HCV infection.
In certain embodiments of the invention, the underlying cause of
the liver disease is HBV infection. Chronic infection with HBV
leads to cirrhosis of the liver and is, with chronic HCV infection,
responsible for making liver cancer the most common cancer in many
parts of the world. Worldwide, around 2 billion people are infected
with HBV. HCC risk is around 20 times higher in people with HBV
and/or HCV infection in Western industrialized countries, where
prevalence of infection is low.
Alternatively, the liver disease may be alcoholic liver disease,
where the underlying cause of the liver disease is alcoholism.
Alcohol intake has been definitely recognized as a cause of chronic
liver diseases, including hepatocellular carcinoma. Alcohol could
be involved in the development of HCC through both direct
(genotoxic) and indirect mechanisms. An indirect mechanism includes
the development of cirrhosis, which is probably the most common
pathway to liver carcinogenesis in developed countries.
In other embodiments of the preset invention, the underlying cause
of the liver disease is non-alcoholic fatty liver disease (NAFLD).
NAFLD is the most common liver disorder in the Western
industrialized countries. It is considered to be the hepatic
manifestation of the metabolic syndrome. Thus, NAFLD tends to
develop in people who are overweight or obese, and/or who have
diabetes, high cholesterol or high triglycerides. For most people,
NAFLD cause no signs and symptoms, and no complications. But in
some people with NAFLD, the fat that accumulates in the liver can
cause inflammation and scarring in the liver that is believed to
result in fibrosis and cirrhosis. This more serious form of NAFLD
is sometimes called non-alcoholic steatohepatitis. It is worth
noting that metabolic syndrome and type 2 diabetes have been
demonstrated to be independent risk factors of HCC.
In yet other embodiments, the underlying cause of the liver disease
is an inherited metabolic disease, such as hereditary
hemochromatosis. People with hereditary hemochromatosis absorb too
much iron from their food. The iron settles in tissues throughout
the body, including the liver. If enough iron builds up in the
liver, it can lead to cirrhosis. Other inherited metabolic diseases
that are risk factors for hepatocellular carcinoma include, alpha 1
antitrypsin deficiency, porphyria cutanea tarda, Wilson's disease,
tyrosinemia, and glycogen storage diseases.
In still other embodiments, the underlying cause of the liver
disease is autoimmune hepatitis (also called lupoid hepatitis).
Autoimmune hepatitis is a chronic disease of the liver that occurs
when the body's immune system attacks cells of the liver causing
the liver to be inflamed. Another autoimmune disease that affects
the liver and can cause cirrhosis is primary biliary cirrhosis or
PBC. PBC is an autoimmune condition, in which the immune system
slowly attacks the bile ducts in the liver. When the bile ducts are
damaged, bile builds up in the liver and over time damages the
tissue. This can lead to scaring, fibrosis and cirrhosis.
In other embodiments, the underlying cause of liver disease is
exposure to aflatoxins. Aflatoxins are poisons produced by a fungus
that grows on crops (such as peanuts, wheat, soybeans, corn, and
rice) that are stored poorly. Long term exposure to these
substances is a major risk for liver cancer. The risk is increased
even more in people with HCV or HBV infection. In developed
countries, the content of aflatoxin in foods is regulated through
testing. Aflatoxin contamination is more common in certain parts of
Africa and Asia.
In still other embodiments, the underlying cause of liver disease
is unknown or the liver disease is caused by yet to be discovered
agents including agents of genetic origin, infectious agents or
chemical and/or physical liver toxic agents.
Administration of an anti-Claudin 1 antibody, or of a
pharmaceutical composition thereof, to patients suffering from
non-HCV associated liver diseases according to the present
invention may slow, reduce, stop or alleviate the progression of
the liver disease, in particular the progression to cirrhosis
and/or to hepatocellular carcinoma, or reverse the progression to
the point of curing the liver disease.
Alternatively or additionally, administration of an anti-Claudin 1
antibody, or of a pharmaceutical composition thereof, to a patient
suffering from a non-HCV associated liver disease according to the
present invention may result in amelioration of at least one of the
symptoms experienced by the individual including, but not limited
to, decreased appetite, weight loss, fatigue, abdominal pain,
jaundice, itching, flu-like symptoms, muscle pain, joint pain,
intermittent low-grade fevers, itching, sleep disturbances, nausea,
diarrhea, dyspepsia, cognitive changes, depression, headaches, and
mood swings; symptoms of cirrhosis such as ascites, bruising and
bleeding tendency, bone pain, varices (especially in the stomach
and esophagus), steatorrhea, jaundice and hepatic
encephalopathy.
Alternatively or additionally, administration of an anti-Claudin 1
antibody, or o a pharmaceutical composition thereof, to a patient
suffering from a non-HCV associated liver disease according to the
present invention may result in prevention of liver
transplantation.
The effects of a treatment according to the invention may be
monitored using any of the assays known in the art for the
diagnosis of the liver disease affecting the patient. Such assays
include, but are not limited to, serological blood tests, and liver
function tests to measure one or more of albumin, alanine
transaminase (ALT), alkaline phosphatase (ALP), aspartate
transaminase (AST), and gamma glutamyl transpeptidase (GGT), and
liver imaging techniques such as magnetic resonance elastography
(MRE), magnetic resonance imaging (MRI), computerized tomography
(CT) and ultrasound. Biopsy may also be performed.
Such assays may also include analysis of liver cell signaling,
transcriptional or proteomic changes, as described in the Examples
below, in a biological sample obtained from the subject receiving a
treatment according to the present invention. Liver cells that can
be analyzed include hepatocytes, Kupffer cells, stellate cells,
endothelial cells, fibroblasts, macrophages, and immune cells
including, but not limited to, T-, B- and NK cells.
In certain embodiments, an anti-Claudin 1 antibody (or a
biologically active fragment thereof) or a pharmaceutical
composition thereof, is administered alone according to a method of
prevention or treatment of the present invention. In other
embodiments, an anti-Claudin 1 antibody (or a biologically active
fragment thereof) or a pharmaceutical composition thereof, is
administered in combination with at least one additional
therapeutic agent. The anti-Claudin 1 antibody (or biologically
active fragment thereof), or pharmaceutical composition thereof,
may be administered prior to administration of the therapeutic
agent, concurrently with the therapeutic agent, and/or following
administration of the therapeutic agent.
Therapeutic agents that may be administered in combination with an
anti-Claudin 1 antibody (or biologically active fragment thereof),
or a pharmaceutical composition thereof, may be selected among a
large variety of biologically active compounds that are known in
the art to have a beneficial effect in the treatment of liver
disease and/or in the treatment of the underlying cause of the
liver disease. As will be understood by one skilled in the art, the
therapeutic agent(s) will differ depending on the nature of the
liver disease that affects the patient.
For example, when the patient is suffering from a liver disease
associated with HBV infection, the therapeutic agent(s) may be
pegylated interferon (PEG-IFN) or nucleoside or nucleotide
analogues that are used in the prevention of HCC in HBV infected
patients. In the case of alcoholic liver disease, the therapeutic
agent(s) may be corticosteroids and/or antioxidants such as
S-adenosyl methionine. When the patient has non-alcoholic fatty
liver disease, the therapeutic agent(s) may be insulin sensitizers
(such as metformin and thiazolidinediones, e.g. Pioglitazone),
ursodeoxycholic acid and lipid-lowering drugs, vitamin E, and
statins. In the case of hereditary hemochromatosis, the therapeutic
agent(s) may be iron chelation drugs (such as chloroquine and
hydroxychloroquine). For alpha 1 antitrypsin deficiency liver
disease, the therapeutic agent(s) may be inhaled forms of alpha 1
antitrypsin. For porphyria cutanea tarda, the therapeutic agent(s)
may be iron chelating drugs (such as chloroquine and
hydroxychloroquine). In the case of Wilson's disease, the
therapeutic agent(s) may be cupper chelating drugs (such as
penicillamine and trientine hydrochloride) and zinc acetate, which
prevent the body from absorbing copper from food. In the case of
autoimmune hepatitis, the therapeutic agent(s) may be
corticosteroids and/or immune system suppressors. For primary
biliary cirrhosis, the therapeutic agent(s) may be ursodeoxycholic
acid (which is the major medication to show the progression of the
disease), immunosuppressive agents, methothrexate, corticosteroids,
cyclosporine and antipruritic agents.
B. Administration
An anti-Claudin 1 antibody, or a biologically active fragment
thereof, (optionally after formulation with one or more appropriate
pharmaceutically acceptable carriers or excipients), in a desired
dosage, can be administered to a subject in need thereof by any
suitable route. Various delivery systems are known and can be used
to administer antibodies, including tablets, capsules, injectable
solutions, encapsulation in liposomes, microparticles,
microcapsules, etc. Methods of administration include, but are not
limited to, dermal, intradermal, intramuscular, intraperitoneal,
intralesional, intravenous, subcutaneous, intranasal, pulmonary,
epidural, ocular, and oral routes. An anti-Claudin 1 antibody, or a
biologically active fragment thereof, or a pharmaceutical
composition thereof, may be administered by any convenient or other
appropriate route, for example, by infusion or bolus injection, by
absorption through epithelial or mucocutaneous linings (e.g., oral,
mucosa, rectal and intestinal mucosa, etc). Administration can be
systemic or local. Parenteral administration may be preferentially
directed to the patient's liver, such as by catheterization to
hepatic arteries or into a bile duct or into the portal vein. As
will be appreciated by those of ordinary skill in the art, in
embodiments where an inventive antibody is administered in
combination with an additional therapeutic agent, the antibody and
therapeutic agent may be administered by the same route (e.g.,
intravenously) or by different routes (e.g., intravenously and
orally).
C. Dosage
An anti-Claudin 1 antibody, or a biologically active fragment
thereof, (optionally after formulation with one or more appropriate
pharmaceutically acceptable carriers or excipients), will be
administered in a dosage such that the amount delivered is
effective for the intended purpose. The route of administration,
formulation and dosage administered will depend upon the
therapeutic effect desired, the severity of the condition to be
treated if already present, the presence of any infection, the age,
sex, weight, and general health condition of the patient as well as
upon the potency, bioavailability, and in vivo half-life of the
antibody or composition used, the use (or not) of concomitant
therapies, and other clinical factors. These factors are readily
determinable by the attending physician in the course of the
therapy. Alternatively or additionally, the dosage to be
administered can be determined from studies using animal models
(e.g., chimpanzee or mice). Adjusting the dose to achieve maximal
efficacy based on these or other methods are well known in the art
and are within the capabilities of trained physicians. As studies
are conducted using anti-Claudin 1 antibodies, further information
will emerge regarding the appropriate dosage levels and duration of
treatment.
A treatment according to the present invention may consist of a
single dose or multiple doses. Thus, administration of an
anti-Claudin 1 antibody, or a biologically active fragment thereof,
(or a pharmaceutical composition thereof) may be constant for a
certain period of time or periodic and at specific intervals, e.g.,
hourly, daily, weekly (or at some other multiple day interval),
monthly, yearly (e.g., in a time release form). Alternatively, the
delivery may occur at multiple times during a given time period,
e.g., two or more times per week; two or more times per month, and
the like. The delivery may be continuous delivery for a period of
time, e.g., intravenous delivery.
In general, the amount of anti-Claudin 1 antibody, or a
biologically active fragment thereof, (or a pharmaceutical
composition thereof) administered will preferably be in the range
of about 1 ng/kg to about 100 mg/kg body weight of the subject, for
example, between about 100 ng/kg and about 50 mg/kg body weight of
the subject; or between about 1 .mu.g/kg and about 10 mg/kg body
weight of the subject, or between about 100 .mu.g/kg and about 1
mg/kg body weight of the subject.
In certain embodiments, the amount of anti-Claudin 1 antibody, or
of a biologically active fragment thereof, (or of a pharmaceutical
composition thereof) administered will be such that the amount
would have no effect on HCV load if it had been administered to a
HCV infected patient.
III--Pharmaceutical Compositions
As mentioned above, anti-Claudin-1 antibodies (and related
molecules) may be administered per se or as a pharmaceutical
composition. Accordingly, the present invention provides
pharmaceutical compositions comprising an effective amount of an
anti-Claudin 1 antibody, or a biologically active fragment thereof,
described herein and at least one pharmaceutically acceptable
carrier or excipient for use in the prevention or treatment of
hepatocellular carcinoma. In some embodiments, the composition
further comprises one or more additional biologically active
agents.
The antibodies or pharmaceutical compositions may be administered
in any amount and using any route of administration effective for
achieving the desired prophylactic and/or therapeutic effect. The
optimal pharmaceutical formulation can be varied depending upon the
route of administration and desired dosage. Such formulations may
influence the physical state, stability, rate of in vivo release,
and rate of in vivo clearance of the administered active
ingredient.
The pharmaceutical compositions of the present invention may be
formulated in dosage unit form for ease of administration and
uniformity of dosage. The expression "unit dosage form", as used
herein, refers to a physically discrete unit of an anti-Claudin-1
antibody, or a biologically active fragment thereof, for the
patient to be treated. It will be understood, however, that the
total daily dosage of the compositions will be decided by the
attending physician within the scope of sound medical
judgement.
A. Formulation
Injectable preparations, for example, sterile injectable aqueous or
oleaginous suspensions may be formulated according to the known art
using suitable dispersing or wetting agents, and suspending agents.
The sterile injectable preparation may also be a sterile injectable
solution, suspension or emulsion in a non-toxic parenterally
acceptable diluent or solvent, for example, as a solution in
2,3-butanediol. Among the acceptable vehicles and solvents that may
be employed are water, Ringer's solution, U.S.P. and isotonic
sodium chloride solution. In addition, sterile, fixed oils are
conventionally employed as a solution or suspending medium. For
this purpose, any bland fixed oil can be employed including
synthetic mono- or di-glycerides. Fatty acids such as oleic acid
may also be used in the preparation of injectable formulations.
Sterile liquid carriers are useful in sterile liquid form
compositions for parenteral administration.
Injectable formulations can be sterilized, for example, by
filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use. Liquid
pharmaceutical compositions which are sterile solutions or
suspensions can be administered by, for example, intravenous,
intramuscular, intraperitoneal or subcutaneous injection. Injection
may be via single push or by gradual infusion. Where necessary or
desired, the composition may include a local anesthetic to ease
pain at the site of injection.
In order to prolong the effect of an active ingredient (here an
anti-Claudin-1 antibody, or a biologically active fragment
thereof), it is often desirable to slow the absorption of the
ingredient from subcutaneous or intramuscular injection. Delaying
absorption of a parenterally administered active ingredient may be
accomplished by dissolving or suspending the ingredient in an oil
vehicle. Injectable depot forms are made by forming
micro-encapsulated matrices of the active ingredient in
biodegradable polymers such as polylactide-polyglycolide. Depending
upon the ratio of active ingredient to polymer and the nature of
the particular polymer employed, the rate of ingredient release can
be controlled. Examples of other biodegradable polymers include
poly(orthoesters) and poly(anhydrides). Depot injectable
formulations can also be prepared by entrapping the active
ingredient in liposomes or microemulsions which are compatible with
body tissues.
Liquid dosage forms for oral administration include, but are not
limited to, pharmaceutically acceptable emulsions, microemulsions,
solutions, suspensions, syrups, elixirs, and pressurized
compositions. In addition to the anti-Claudin-1 antibody, or
biologically active fragment thereof, the liquid dosage form may
contain inert diluents commonly used in the art such as, for
example, water or other solvent, solubilising agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in
particular, cotton seed, ground nut, corn, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols, and fatty acid esters of sorbitan and mixtures thereof.
Besides inert diluents, the oral compositions can also include
adjuvants such as wetting agents, suspending agents, preservatives,
sweetening, flavouring, and perfuming agents, thickening agents,
colors, viscosity regulators, stabilizes or osmo-regulators.
Examples of suitable liquid carriers for oral administration
include water (potentially containing additives as above, e.g.,
cellulose derivatives, such as sodium carboxymethyl cellulose
solution), alcohols (including monohydric alcohols and polyhydric
alcohols such as glycols) and their derivatives, and oils (e.g.,
fractionated coconut oil and arachis oil). For pressurized
compositions, the liquid carrier can be halogenated hydrocarbon or
other pharmaceutically acceptable propellant.
Solid dosage forms for oral administration include, for example,
capsules, tablets, pills, powders, and granules. In such solid
dosage forms, an anti-Claudin-1 antibody, or a biologically active
fragment thereof may be mixed with at least one inert,
physiologically acceptable excipient or carrier such as sodium
citrate or dicalcium phosphate and one or more of: (a) fillers or
extenders such as starches, lactose, sucrose, glucose, mannitol,
and silicic acid; (b) binders such as, for example,
carboxymethylcellulose, alginates, gelatine, polyvinylpyrrolidone,
sucrose, and acacia; (c) humectants such as glycerol; (d)
disintegrating agents such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate; (e) solution retarding agents such as paraffin;
absorption accelerators such as quaternary ammonium compounds; (g)
wetting agents such as, for example, cetyl alcohol and glycerol
monostearate; (h) absorbents such as kaolin and bentonite clay; and
(i) lubricants such as talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulphate, and mixtures
thereof. Other excipients suitable for solid formulations include
surface modifying agents such as non-ionic and anionic surface
modifying agents. Representative examples of surface modifying
agents include, but are not limited to, poloxamer 188, benzalkonium
chloride, calcium stearate, cetostearyl alcohol, cetomacrogol
emulsifying wax, sorbitan esters, colloidal silicon dioxide,
phosphates, sodium dodecylsulfate, magnesium aluminum silicate, and
triethanolamine. In the case of capsules, tablets and pills, the
dosage form may also comprise buffering agents.
Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatine capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like. The solid dosage forms of
tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings, release
controlling coatings and other coatings well known in the
pharmaceutical formulating art. They may optionally contain
opacifying agents and can also be of a composition such that they
release the active ingredient(s) only, or preferably, in a certain
part of the intestinal tract, optionally, in a delaying manner.
Examples of embedding compositions which can be used include
polymeric substances and waxes.
In certain embodiments, it may be desirable to administer an
inventive composition locally to an area in need of treatment
(e.g., the liver). This may be achieved, for example, and not by
way of limitation, by local infusion during surgery (e.g., liver
transplant), topical application, by injection, by means of a
catheter, by means of suppository, or by means of a skin patch or
stent or other implant.
For topical administration, the composition is preferably
formulated as a gel, an ointment, a lotion, or a cream which can
include carriers such as water, glycerol, alcohol, propylene
glycol, fatty alcohols, triglycerides, fatty acid esters, or
mineral oil. Other topical carriers include liquid petroleum,
isopropyl palmitate, polyethylene glycol, ethanol (95%),
polyoxyethylenemonolaurat (5%) in water, or sodium lauryl sulphate
(5%) in water. Other materials such as antioxidants, humectants,
viscosity stabilizers, and similar agents may be added as
necessary.
In addition, in certain instances, it is expected that the
pharmaceutical compositions may be disposed within transdermal
devices placed upon, in, or under the skin. Such devices include
patches, implants, and injections which release the active
ingredient by either passive or active release mechanisms.
Transdermal administrations include all administration across the
surface of the body and the inner linings of bodily passage
including epithelial and mucosal tissues. Such administrations may
be carried out using the present compositions in lotions, creams,
foams, patches, suspensions, solutions, and suppositories (rectal
and vaginal).
Transdermal administration may be accomplished through the use of a
transdermal patch containing an active ingredient (i.e., an
anti-Claudin-1 antibody, or a biologically active fragment thereof)
and a carrier that is non-toxic to the skin, and allows the
delivery of the ingredient for systemic absorption into the
bloodstream via the skin. The carrier may take any number of forms
such as creams and ointments, pastes, gels, and occlusive devices.
The creams and ointments may be viscous liquid or semisolid
emulsions of either the oil-in-water or water-in-oil type. Pastes
comprised of absorptive powders dispersed in petroleum or
hydrophilic petroleum containing the active ingredient may be
suitable. A variety of occlusive devices may be used to release the
active ingredient into the bloodstream such as a semi-permeable
membrane covering a reservoir containing the active ingredient with
or without a carrier, or a matrix containing the active
ingredient.
Suppository formulations may be made from traditional materials,
including cocoa butter, with or without the addition of waxes to
alter the suppository's melting point, and glycerine. Water soluble
suppository bases, such as polyethylene glycols of various
molecular weights, may also be used.
Materials and methods for producing various formulations are known
in the art and may be adapted for practicing the subject invention.
Suitable formulations for the delivery of antibodies can be found,
for example, in "Remington's Pharmaceutical Sciences", E. W.
Martin, 18.sup.th Ed., 1990, Mack Publishing Co.: Easton, Pa.
B. Additional Biologically Active Agents
In certain embodiments, an anti-Claudin-1 antibody, or a
biologically active fragment thereof, is the only active ingredient
in a pharmaceutical composition of the present invention. In other
embodiments, the pharmaceutical composition further comprises one
or more biologically active agents. Examples of suitable
biologically active agents include, but are not limited to,
therapeutic agents such as anti-viral agents, anti-inflammatory
agents, immunomodulatory agents, analgesics, antimicrobial agents,
kinase inhibitors, signalling inhibitors, antibacterial agents,
antibiotics, antioxidants, antiseptic agents, and combinations
thereof.
In such pharmaceutical compositions, the anti-Claudin-1 antibody
and additional therapeutic agent(s) may be combined in one or more
preparations for simultaneous, separate or sequential
administration of the anti-Claudin-1 antibody and therapeutic
agent(s). More specifically, an inventive composition may be
formulated in such a way that the antibody and therapeutic agent(s)
can be administered together or independently from each other. For
example, an anti-Claudin-1 antibody and a therapeutic agent can be
formulated together in a single composition. Alternatively, they
may be maintained (e.g., in different compositions and/or
containers) and administered separately.
C. Pharmaceutical Packs of Kits
In another aspect, the present invention provides a pharmaceutical
pack or kit comprising one or more containers (e.g., vials,
ampoules, test tubes, flasks or bottles) containing one or more
ingredients of an inventive pharmaceutical composition, allowing
administration of an anti-Claudin-1 antibody, or a biologically
active fragment thereof.
Different ingredients of a pharmaceutical pack or kit may be
supplied in a solid (e.g., lyophilized) or liquid form. Each
ingredient will generally be suitable as aliquoted in its
respective container or provided in a concentrated form.
Pharmaceutical packs or kits may include media for the
reconstitution of lyophilized ingredients. Individual containers of
the kits will preferably be maintained in close confinement for
commercial sale.
In certain embodiments, a pharmaceutical pack or kit includes one
or more additional therapeutic agent(s) as described above.
Optionally associated with the container(s) can be a notice or
package insert in the form prescribed by a governmental agency
regulating the manufacture, use or sale of pharmaceutical or
biological products, which notice reflects approval by the agency
of manufacture, use or sale for human administration. The notice of
package insert may contain instructions for use of a pharmaceutical
composition according to methods of treatment disclosed herein.
An identifier, e.g., a bar code, radio frequency, ID tags, etc.,
may be present in or on the kit. The identifier can be used, for
example, to uniquely identify the kit for purposes of quality
control, inventory control, tracking movement between workstations,
etc.
EXAMPLES
The following examples describe some of the preferred modes of
making and practicing the present invention. However, it should be
understood that the examples are for illustrative purposes only and
are not meant to limit the scope of the invention. Furthermore,
unless the description in an Example is presented in the past
tense, the text, like the rest of the specification, is not
intended to suggest that experiments were actually performed or
data are actually obtained.
Some of the results reported below are presented in a manuscript:
T. Baumert et al., "A Claudin-1-specific Monoclonal Antibody for
Prevention and Treatment of Hepatocellular Carcinoma", which has
been submitted for publication.
Example 1
Materials and Methods
Reagents and Antibodies.
The anti-claudin-1 monoclonal antibodies (anti-CLDN1 mAbs were
produced as previously described (Fofana et al., Gastroenterology,
2010, 139: 953-964, e1-4). Erlotinib was purchased from IC
Laboratories; and interferon-alpha 2a from Roche. Daclatasvir and
sofosbuvir were synthesized by Acme Biosciences. Pioglitazone,
metformin and DMSO were purchased from Sigma-Aldrich. The Human
Phospho-RTK Array kit was obtained from R&D Systems. The ECL
reagent and Hyperfilms were purchased from GE Healthcare. The
Alexa-Fluor.RTM. 647 anti-mouse IgG (goat) and Alexa-Fluor.RTM. 647
anti-human IgG (goat) were purchased from Jackson ImmunoResearch.
Dapi was obtained from Life Technologies.
Cell Lines.
Huh7.5.1 cells have already been described (Zhong et al., Proc Natl
Acad Sci USA, 2005, 102(26): 9294-9299). For proliferation arrest
and differentiation (Huh7.5.1.sup.dif cells), 2.5.10.sup.4 to
3.10.sup.4 Huh7.5.1 cells were cultured in Dulbecco's Modified
Eagle Medium (DMEM) containing 1% dimethylsulfoxide (DMSO).
HCV Infection of Huh7.5.1.sup.dif Cells.
Cell culture-derived HCVcc Jc1 (genotype 2a/2a) (Pietschmann et
al., Proc Natl Acad Sci USA, 2006, 103(19): 7408-7413) were
generated in Huh7.5.1 cells as previously described (Wakita et al.,
Nat Med., 2005, 11(7): 791-796). HCVcc infectivity was determined
by calculating the 50% tissue culture infectious doses
(TCID.sub.50) in infection experiments as previously described
(Lindenbach et al., Science, 2005, 309(5734): 623-626). HCV
infection was assessed by qRT-PCR of intracellular HCV RNA (Xiao et
al., Gut, 2015, 64(3): 483-494) as well as by immunostaining using
an HCV E2-specific AP33 antibody as previously described (Krieger
et al., Hepatology. 2010, 51(4): 1144-1157).
Transcriptional Analyses.
Liver cells were lysed in TRI-reagent (Molecular Research Center),
and RNA was purified using Direct-zol RNA MiniPrep (Zymo Research)
according to the manufacturer's instructions. RNA quantity and
quality were assessed using NanoDrop (ThermoScientific) and
Bioanalyzer 2100 (Illumina). Gene expression profiling was
performed using 250-500 ng total RNA by using nCounter Digital
Analyzer system (NanoString).
Analysis of Phosphorylation.
Phospho-array analysis was performed using the Proteome Prolifer
Human Phospho-kinase Array (R&D Systems) as previously
described by the manufacturer. For imaging, blots were incubated
with ECL (GE Healthcare) and exposed to ECL Hyperfilm (GE
Healthcare). Phospho-kinase array results were quantified by
integrating the dot blot densities using Image J software
(NIH).
Effect of Antivirals and Small Molecules on HCC Risk Signature.
Seven (7) days after HCV Jc1 infection, Huh7.5.1.sup.dif cells were
incubated with either a combination of 1 nM daclatasvir and 1 .mu.M
sofosbuvir; 10 IU/mL interferon-alpha 2a; 1, 10 or 100 .mu.g/mL
CLDN1-specific mAb; or 0.1 mM erlotinib in the presence of 1% DMSO.
Cells incubated with 1% DMSO served as negative control. Three (3)
days after treatment, the cells were lysed, total RNA was purified
as described above, and analyzed for gene expression and
intracellular viral load as described above.
Bioinformatic and Statistical Analyses.
Prediction of clinical outcome based on the 186-gene signature was
performed as previously reported by using the nearest template
prediction algorithm, which was implemented in GenePattern (King et
al., Gut, 2014, 20. pii: gutjn1-2014-307862. doi:
10.1136/gutjn1-2014-307862; Hoshida et al., PLoS ONE, 2010, 5(11),
e15543). A prediction of HCC high-risk or low-risk gene signature
was determined using p<0.05 and FDR<0.25.
Induction/suppression of each gene set over time according to HCV
Jc1 infection and uninfected control was assessed through GSEA and
single sample GSEA (ssGSEA) modules implemented in GenePattern as
previously described (Subramanian et al., Proc Natl Acad Sci USA,
2005, 102(43): 15545-15550; Barbie et al., Nature, 2009, 462(7269):
108-112). Pathway enrichment analysis was performed using ToppGene
Suite (webpage: toppgene.cchmc.org) as previously described (Chen
et al., Nucleic Acids Res., 2009, 37(Web Server issue): W305-311).
nCounter assay genes were considered significantly expressed with
t-test FDR<0.05 and fold changes of .+-.1.8. Using
differentially expressed genes, network analysis was performed
using canonical pathways from Ingenuity Knowledge Base repository
(Ingenuity Systems Inc.).
Results
186-Gene HCC Risk Signature.
The present study makes use of a 186-gene HCC risk signature in
non-cancerous liver tissue, which has been shown to be strongly
associated with HCC predictive risk in patients with cirrhosis
caused by hepatitis C virus (HCV), hepatitis B virus (HBV) and
alcoholism (Hoshida et al., N Engl J Med., 2008, 359(19):
1995-2004; Hoshida et al., Gastroenterology. 2013, 144(5):
1024-1030; King et al., Gut, 2014, 20. pii: gutjn1-2014-307862.
doi: 10.1136/gutjn1-2014-307862; King et al., PLoS ONE, 2014,
9(12): e114747) in multiple independent cohorts of Asian, European,
and American patients with cirrhosis and HCC caused by multiple
etiologies based on up to 23 years of follow-up. This gene
signature comprised 73 HCC high-risk genes in liver, which were
up-regulated, and 103 HCC low-risk genes, which were down-regulated
in liver tissues (Hoshida et al., Gastroenterology. 2013, 144(5):
1024-1030).
Molecular pathway analysis had revealed that chronic HCV infection
in the human liver enhances activation of NF-.kappa.B,
interleukin-6, as well as epidermal growth factor (EGF) pathways
and suppresses DNA repair-related genes (Hernandez-Gea et al.,
Gastroenterology, 2013, 144(3): 512-527). The signature
demonstrated substantially superior prognostic capability (Hoshida
et al., J Hepatol., 2014, 61(1S): S79-S90) compared to prognostic
DNA variants identified in large-scale case-control or cohort
studies (Kumar et al., Nat Genet., 2011, 43(5): 455-458; Miki et
al., Nat Genet., 2011, 43(8): 797-800; Abu Dayyeh et al.,
Gastroenterology, 2011, 141(1): 141-149). In rodent models of
cirrhosis-driven HCC, the signature was induced from a very early
stage of liver fibrosis, and reversed in response to the
FDA-approved EGF pathway inhibitor erlotinib, accompanied with
reduced liver fibrosis and HCC nodules (Fuchs et al., Hepatology,
2014, 59(4): 1577-1590).
Thus, the 186-gene HCC risk signature represents a valuable tool to
monitor progression of HCC and to understand what pathways play a
role in HCC development. Taking advantage of this observation, the
present Applicants have recently developed a simple and robust
liver cell-based system with inducible 186-gene HCC risk signature.
The HCC high-risk signature was induced by persistent HBV
infection, HCV infection or ethanol exposure. Using this model,
they have identified drivers of the HCC high-risk signature,
including EGFR signaling, and showed reversal of the HCC high-risk
signature by erlotinib (S. Bandiera et al., "A cell-based model
unravels drivers for hepatocarcinogenesis and targets for clinical
chemoprevention", which was submitted for publication to Nature
Medicine on Mar. 12, 2015. This cell-based model enables to unravel
the cell circuits of liver disease progression in patients and to
identify HCC chemoprevention targets for clinical evaluation.
A CLDN1-Specific mAb was Found to Reverse the HCC High-Risk Gene
Expression Signature in a Liver Cell-Based Model for Liver Disease
Progression and Hepatocarcinogenesis.
To evaluate the role of the anti-CLDN1 mAb for HCC chemoprevention,
the Applicants used a previously developed model based on poorly
proliferative Huh7.5.1 cells (Huh7.5.1.sup.dif) in which the
HCC-related 186-gene signature can be readily induced upon exposure
to recombinant HCV (strain Jc1--Bauhofer et al., Gastroenterology,
2012, 143(2): 429-438 e8) (see FIG. 1A). The CLDN1-specific mAb was
added to Huh7.5.1.sup.dif cells 7 days after viral inoculation, a
time point at which the vast majority of cells was persistently
infected with HCV as assessed by immunocytochemical assay (see FIG.
1B). Ertolinib was used as a positive control for reversal of the
gene signature. The effect of the CLDN1-specific mAb treatment on
the expression of the HCV-induced HCC-related 186-gene signature
was then assessed using digital transcript counting technology
(nCounter assay) (Hoshida et al., N Engl J Med., 2008, 359(19):
1995-2004; King et al., Gut, 2014, 20. pii: gutjn1-2014-307862.
doi: 10.1136/gutjn1-2014-307862). Gene Set Enrichment Analysis
(GSEA) indicated that HCC high-risk genes were potently suppressed
(NES: -2.29, FDR<0.001), and HCC low risk genes were
significantly induced (NES: 1.73, FDR<0.001) in CLDN1-specific
mAb-treated samples. Furthermore, using a computational analysis
using a HCC prediction model that applies a nearest template
algorithm (Hoshida et al., PLoS ONE, 2010, 5(11): e15543), the
Applicants demonstrated a statistically significant reversal of the
186-gene signature in all persistently HCV-infected samples after
CLDN1-specific mAb treatment (see Table 1 below).
TABLE-US-00001 TABLE 1 HCC prediction model showed the reversal of
HCC-high risk genes following CLDN1-specific mAb. Huh7.5.1.sup.dif
cells were HCV Jc1 infected. Total cellular RNA was isolated and
subjected to NanoString analysis. Gene expression data was
submitted to Nearest template prediction (NTP) of 186-gene-HCC risk
signature in HCV infected Huh7.5.1.sup.dif cells treated with 10
and 100 .mu.g/ml of CLDN1 specific mAb, analysis was performed
using GenePattern. Heatmaps were obtained (data not shown).
HCV-infected (Ctrl) and treated (CLDN1_10 or CLDN1_100) cells were
predicted as HCC high risk and low risk. NTP statistical figures
are presented below. FDR p-values < 0.05 were considered
significant. 10 .mu.g/mL 100 .mu.g/mL Sample p-value Sample p-value
CTRL_10_3 0.0005 CTRL_100_3 0.0005 CTRL_10_2 0.0005 CTRL_100_2
0.0005 CTRL_10_1 0.008 CTRL_100_1 0.0075 CLDN1_10_2 0.0005
CLDN1_100_2 0.004 CLDN1_10_3 0.0005 CLDN1_100_1 0.0055 CLDN1_10_1
0.0005 CLDN1_100_3 0.001
The CLDN1-Specific mAb was Found to Reverse the 186-Gene HCC Risk
Signature in Cells More Potently than Direct-Acting Antivirals and
than HCC Chemopreventive Candidate Compounds.
Strikingly, the effect of the CLDN1-specific mAb was more potent
than the effect of erlotinib which induced partial suppression of
HCC high-risk genes (NES: -1.28, FDR=0.08) and induction of HCC
low-risk genes (NES: 1.26, FDR=0.09) (see FIG. 1C).
To further assess the potency of the CLDN1-specific mAb in
reversing the HCC risk gene signature, the Applicants compared its
effects to those of direct-acting antivirals (DAAs), interferon
alpha and pioglitazone, compounds that the present Applicants have
previously shown to partially suppress the HCC high-risk genes
using GSEA. Metformin, a compound that was previously shown not to
exhibit any effect on the HCC risk signature, was used as a
negative control. The present Applicants found that the
CLDN1-specific mAb reverses HCC high-risk genes more potently than
the other tested compounds (see FIG. 2A). Notably, in contrast to
DAAs, CLDN1-specific mAb-induced suppression of the HCC high-risk
genes appeared to be independent from its effect on viral load
since very low concentrations of this monoclonal antibody that do
not modulate viral load in Huh7.5.1.sup.dif cells were potent in
reversing the 186-gene signature (see FIG. 2B).
Taken together, these data indicate that the CLDN1-specific mAb
potently reverses the HCC risk signature induced by HCV
independently of its antiviral effect.
The CLDN1-Specific mAb was Found to Impair EGF-MAPK Signalling as a
Driver for Hepatocarcinogenesis.
The Applicants have previously shown that HCV uses EGFR and
Claudin-1 as host dependency factors to enter the hepatocyte in
cell culture and in vivo (Mailly et al., Clearance of persistent
hepatitis C virus infection using a claudin-1-targeting monoclonal
antibody, Nat Biotech, 2015, 33(5): 549-554; Lupberger et al.,
Hepatology, 2013, 58(4): 1225-1235; Lupberger et al., Nat Med.,
2011, 17(5): 589-559; Zona et al., Cell Host Microbe, 2013, 13(3):
302-313). To assess whether HCV not only exploits EGFR and CLDN1
for entry, but also triggers intracellular signaling cascades, the
present Applicants investigated virus-induced signaling in
virus-infected liver cells. Taking advantage of the novel model
system to study HCV disease biology (see FIG. 1), they screened the
activation state of hepatocyte canonical signaling pathways, as
previously described (Lupberger et al., Hepatology, 2013, 58(4):
1225-1235; Lupberger et al., Nat Med., 2011, 17(5): 589-59; Zona et
al., Cell Host Microbe, 2013, 13(3): 302-313). They observed that
HCV infection triggers activation of specific host signaling
networks, including EGFR pathway as shown by virus-induced EGFR
phosphorylation (see FIG. 3A-B), enhanced EGF and EGFR expression
and significant induction of experimentally-defined EGF target gene
signatures (see FIG. 3C). Interestingly, an induction of the
EGF/EGFR pathway was also observed in HBV-infected as well as in
ethanol-treated cells, albeit at a lower level (see FIG. 3D-E).
Analysis of downstream signaling pathways revealed HCV-induced
phosphorylation of extracellular signal-regulated kinase (ERK) (see
FIG. 3F). Since the EGFR pathway has been identified to be strongly
associated with hepatocarcinogenesis in patients (Hoshida et al., N
Engl J Med., 2008, 359(19):1995-2004; Hoshida et al.,
Gastroenterology, 2013, 144(5):1024-1030; King et al., Gut, 2014,
20. pii: gutjn1-2014-307862. doi: 10.1136/gutjn1-2014-307862; King
et al., PLoS ONE, 2014, 9(12): e114747; Abu Dayyeh et al.,
Gastroenterology, 2011, 141(1): 141-149) and a driver for HCC in
animal models (Fuchs et al., Hepatology, 2014, 59(4): 1577-1590;
Lanaya et al., Nat Cell Biol., 2014, 16(10): 972-981, 1-7), it is
likely that the virus-induced activation of EGFR-ERK1/2 pathways
contributes to hepatocarcinogenesis. Importantly, perturbation data
show that the anti-Claudin 1 mAb inhibits ERK1/2 signaling (see
FIG. 3F). Significant suppression of gene expression of the EGFR
pathway was also observed by analysis of the EGFR oncogenic
signatures in cancer (see FIG. 3G). Single sample GSEA (ssGSEA)
showed the suppression of EGFR signaling and EGFR-related genes
even at low doses of the CLDN1-specific antibody (data not shown).
The involvement of the EGFR pathway was further supported by
network analysis of differentially expressed genes showing the
suppression of EGFR (see FIG. 4A) and MAPK signaling pathways (see
FIG. 4B).
The CLDN1-Specific mAb was Found to Impair Expression of Genes
Related to Liver Disease as Well as Inflammatory Response Genes
which are Drivers of Hepatocarcinogenesis.
Furthermore, functional enrichment analysis of differentially
expressed genes showed that inflammatory responses genes, including
NF-.kappa.B, EBV, LMP1, MyD88 and TLR signaling, were
down-regulated as shown in part A of Table 2 below) and FIG. 5,
while the expression of genes involved in metastatic pathways was
up-regulated (see part B of Table 2 below).
TABLE-US-00002 TABLE 2 CLDN1-specific mAb treatment suppresses
inflammatory-related genes and induces metabolic-related genes.
Huh7.5.1.sup.dif cells were HCV Jc1 infected. Following isolation
of total RNA there were subjected to NanoString analysis. Intensity
expression values were normalized and log transformed.
Differentially expressed genes have FDR p-values < 0.05 and fold
change of .+-. 1.9. Pathway analysis was performed using ToppGene
Suite A. Down-regulated genes belong to MAPK, NF-.kappa.B, and
Toll-like receptor signaling, while B. up-regulated genes belong to
metabolism-related pathways. p-value FDR q-value A. Pathways of
down-regulated genes Induction of NFkB and MAP kinases 8.75E-05
2.02E-02 MyD88 dependent cascade 9.81E-05 2.02E-02 Toll Like
Receptor 7/8 (TLR7/8) Cascade 9.81E-05 2.02E-02 Toll Like Receptor
9 (TLR9) Cascade 1.22E-04 2.02E-02 Toll Like Receptor 3 (TLR3)
Cascade 1.83E-04 2.02E-02 EBV LMP1 signaling 2.24E-04 2.16E-02 CREB
phosphorylation 6.11E-04 4.28E-02 B. Pathways of up-regulated genes
Respiratory electron transport 5.77E-09 2.06E-06 The citric acid
(TCA) cycle 7.50E-09 2.06E-06 Metabolism of proteins 1.11E-05
5.57E-04 Regulation of complement cascade 3.01E-04 1.10E-02
Proteasome Complex 6.23E-04 2.14E-02 Purine nucleotide salvage
1.66E-03 4.14E-02 Purine nucleotides de novo biosynthesis 1.66E-03
4.14E-02
Interestingly, eight (8) of the nine (9) genes that were previously
shown to be induced by different HCC drivers, including HCV, HBV or
ethanol treatment (see Bandiera et al., "A cell-based model
unravels drivers for hepatocarcinogenesis and target for clinical
chemoprevention", which was submitted to Nature Medicine for
publication on Mar. 12, 2015 by the Applicants), were suppressed
following CLDN1-specific mAb treatment (see Table 3 below).
Finally, the present Applicants showed that the Claudin-1 specific
antibody suppresses expression of genes involved in liver diseases
(see FIG. 6).
TABLE-US-00003 TABLE 3 CLDN1-specific mAb treatment down-regulates
genes commonly induced by different HCC drivers including HCV and
HBV infection and ethanol treatment. Statistical figures, FDR
p-values < 0.05 were considered significant. Gene FDR Fold
Symbol Gene Name p-value changes ANXA3 Annexin A3 0.001 -1.92
FILIP1L Filamin A interacting protein 1-like 0.0014 -1.92 DUSP5
Dual specificity phosphatase 5 0.002 -1.94 ANXA1 Annexin A1 0.005
-1.93 EGF Epidermal growth factor 0.005 -1.94 SLC12A2 Solute
carrier family 12 (sodium/ 0.006 -1.95 potassium/chloride
transporter), member 2 PODXL Podocalyxin-like 0.022 -1.95 LOXL2
Lysyl oxidase-like 2 0.036 -1.93
Collectively, these data indicate that the CLDN1-specific mAb
reverses the risk for hepatocarcinogenesis, likely by impairing
EGF-MAPK signaling and expression of inflammatory response
genes.
Discussion
In the present study, the Applicants have shown that CLDN1, a well
characterized HCV entry factor and antiviral target to prevent and
treat HCV infection, is a previously undiscovered target for HCC
prevention and treatment. They demonstrated that a CLDN1-specific
mAb reverses HCC high-risk genes in a liver cell-based model system
where a HCC risk signature common to different etiologies was
induced by persistent HCV infection. Notably, the CLDN1-specific
monoclonal antibody reversed this HCC risk signature more potently
than any other antivirals (direct acting anti-viral, interferon
alpha) or potential HCC chemopreventive agents (erlotinib,
pioglitazone) that they tested. Interestingly, the CLDN1-specific
mAb was found to be able to reverse the HCC risk signature at
concentrations that do not have an effect on HCV load,
demonstrating that the CLDN1-specific mAb may prevent the
progression of HCC independently of its effect as an antiviral
agent and thus exhibit a broad HCC chemoprotective activity
independently of the underlying etiology. Indeed, the present
Applicants have shown that the CLDN1-specific mAb reverses the
expression of 8 out of 9 genes commonly induced by different HCC
etiologies with the exception of GPX2. Collectively, these data
highlight the promise of CLDN1 as a target for HCC
chemoprevention.
CLDN1 expression increases during HCC, in particular in cirrhotic
liver patients (Stebbing et al., Oncogene, 2013, 32(41): 4871-4872)
and it has been shown to contribute to epithelial-mesenchymal
transition (EMT), an early step in tumor progression. The
underlying molecular mechanism may involve the intracellular
signaling cascades downstream of CLDN1 mainly via the activation of
c-Abl-PKC-ERK1/2 axis which promotes epithelial mesenchymal
transition (EMT) through the activation of MMP-2 (Suh et al.,
Oncogene, 2013, 32(41):4873-4882; Yoon et al., J Biol Chem., 2010,
285(1): 226-233). Indeed, in the present study, the Applicants
showed that the CLDN1-specific mAb impairs EGFR/MAPK signalling
(see FIG. 3F,G), a driver for hepatocarcinogenesis (Fuchs et al.,
Hepatology, 2014, 59(4): 1577-1590; Lanaya et al., Nat Cell Biol.,
2014, 16(10): 972-981, 1-7). Complementary to these observations,
the functional enrichment analysis showed that the CLDN1-specific
mAb suppressed expression of pathways related to EGF (see FIG. 4A)
and MAPK signalling (see FIG. 4B). Moreover, the inflammatory
pathway of NF-.kappa.B was shown to be suppressed (see FIG. 5).
This pathway plays an important role in aggravating liver injury by
promoting inflammatory responses in fibrotic livers and promotes
the progression into HCC and metastasis at a later stage (Ning et
al., Hepatology, 2014, 60(5): 1607-1619; Shen et al., Hepatology,
2014, 60(6): 2065-2076; Song et al., Hepatology, 2014, 60(5):
1659-1673). Furthermore, several pathways belonging to MyD88
signaling, a major player in Toll-like receptor signaling, as well
as TLR7 and 9 pathways were down-regulated after CLDN1-specific mAb
treatment (see Part A of Table 1 above). Recently, these pathways
were suggested to be potential HCC therapeutic targets, as they
play a role in inducing inflammatory responses during HCC (Leake et
al., Nat Rev Gastroenterol Hepatol., 2014, 11(9): 518; Mohamed et
al., Liver Int., 2015, 64(3): 483-494).
Taking into account the ability of CLDN1-specific mAb to reverse
signaling (see FIGS. 1-3) and inflammatory responses driving
hepatocarcinogenesis (see FIGS. 3 to 5) (Fuchs et al., Hepatology,
2014, 59(4):1577-1590; Suh et al., Oncogene, 2013, 32(41):
4873-4882; Stebbing et al., Oncogene, 2013; 32(41):4871-4872; Song
et al., Hepatology, 2014, 60(5): 1659-1673; Fortier et al., J Biol
Chem., 2013, 288(16): 11555-11571) as well as suppressing the well
characterized HCC risk gene signature (see FIG. 2) associated with
progression of liver disease (Hoshida et al., N Engl J Med., 2008,
359(19): 1995-2004; Hoshida et al., Gastroenterology, 2013,
144(5):1024-1030; King et al., Gut, 2014, 20. pii:
gutjn1-2014-307862. doi: 10.1136/gutjn1-2014-307862; King et al.,
PLoS ONE, 2014, 9(12): e114747; Abu Dayyeh et al.,
Gastroenterology, 2011, 141(1): 141-149; Fuchs et al., Hepatology,
2014, 59(4): 1577-1590), the CLDN1-specific mAb is suitable for
prevention and treatment of HCC. Given its ability to reverse the
HCC risk gene signature independently of the etiology and
independently of its antiviral effect opens perspectives to utilise
CLDN1-specific mAbs to prevent and treat HCC irrespective of the
etiologic cause including patients with cured HCV infection and
patients with HCC due to chronic hepatitis B virus infection,
alcohol, non-alcoholic fatty liver disease (NAFLD) or autoimmune or
hereditary liver disease.
Example 2
Materials and Methods
Cell Lines.
Cells from the Huh7.5.1 cell line (Zhong et al., Proc Natl Acad Sci
USA, 2005, 102(26): 9294-9299) were cultured in Dulbecco's Modified
Eagle Medium (DMEM) containing 1% dimethylsulfoxide (DMSO) for
differentiation (Huh7.5.1.sup.dif). NTCP-overexpressing HepG2 cells
(HepG2-NTCP) were selected using puromycin and cultured in DMEM (Ni
et al., Gastroenterology, 2014, 146: 1070-1083; Yan et al., eLife,
2012; 1:e00049).
HCV Infection and CLDN1-Specific mAb Treatment.
Huh7.5.1.sup.dif cells were plated in 6-well plates and infected
with HCVcc Jc1 (genotype 2a/2a) (Pietschmann et al., PNAS USA,
2006, 103: 7408-7413; Wakita et al., Nature medicine. 2005, 11(7):
791-796). CLDN1-specific mAb or control Ab (10 .mu.g/ml) was added
at day 7 post-infection. HCV infection was assessed at day 10 by
qRT-PCR of intracellular RNA as previously described (Xiaa et al.,
PLoS pathogens. 2014, 10(5): e1004128).
HBV Infection and CLDN1-Specific mAb Treatment.
HepG2-NTCP cells were plated in 12-well plates and infected with
either recombinant HBV (strain ayw, genotype D) (Ladner et al.,
Antimicrobial agents and chemotherapy, 1997, 41(8): 1715-20) or
serum-purified HBV (Habersetzer et al., Liver international:
Official Journal of the International Association for the Study of
the Liver, 2015, 35(1): 130-139). Human CLDN1-specific mAb or
control Ab (10 .mu.g/ml) was added for 7 days. Rat CLDN1-specific
mAb or control Ab (10 .mu.g/ml) was added for 3 days following 7
days of infection. HBV infection was assessed at day 7 or 10
post-infection by qRT-PCR quantification of HBV pregenomic RNA
(pgRNA) as previously described (Verrier et al., Hepatology, 2016,
63(1): 35-48).
Ethanol Treatment.
Huh7.5.1.sup.dif cells were plated in 6-well plates and exposed to
ethanol (40 mM) and treated with CLDN1-specific or control Ab (10
.mu.g/ml) for 10 days. Fresh medium containing ethanol and
antibodies was replenished daily (Ye et al., Drug and alcohol
dependence, 2010, 112(1-2): 107-116).
Transcriptional Analyses.
See Examples 1 for details. Expression of the HCC-risk signature
gene expression was analyzed using Biomark HD, high-throughput
RT-PCR technology (Baker et al., Nature Med., 2012, 9(6): 541-544).
The expression of EMT-regulated genes was assessed by qRT-PCR
Taqman Gene Expression assays (Life Technologies, USA). Expression
levels were normalized to GAPDG. Relative expression was calculated
using .DELTA..DELTA.Ct method.
Bioinformatic and Statistical Analyses.
Induction/suppression of high-risk or low-risk gene signature
control was assessed through GSEA with FDR<0.25 or using
enrichment scores (ES) for individual genes and normalized
enrichment scores (NES) for gene sets (Subramanian et al., PNAS
USA, 20005, 102(43): 15545-15550).
Metabolomics.
HCV-infected Huh7.5.1.sup.dif cells were treated with
CLDN1-specific mAb on day 7 post-infection. On day 10
post-infection, metabolites were extracted and analyzed by mass
spectrometry. Data was analyzed using MetaboAnalyst 3.0 (Xia et
al., Nucleic acids Research, 2015, 43(W1): W251-W257).
Results
CLDN1-Specific mAbs Reverse the Patient-Derived Panetiology 32-Gene
HCC Risk Signature in HBV-Infected Liver Cell-Based Model.
To assess the potential of the anti-CLDN1 mAbs for prevention of
HCC induced by other etiologies than HCV infection, the Applicants
next assessed their ability to reverse HCC-risk genes modulated by
HBV infection. Recently, a 32-gene signature derived from the
previously described 186-gene HCC-risk signature has been shown to
have the highest significance for prediction of liver disease
progression and HCC development in all major HCC etiologies (HCV,
HBV, alcohol and NASH) (King et al., Gut, 2014, 64(8): 1296-1302).
HepG2 liver-derived cells over-expressing NTCP, a cell entry factor
for HBV, were infected with HBV and treated with human
CLDN1-specific or control mAbs for 7 days (FIG. 7A). Treatment with
the human CLDN1-specific mAb resulted in the induction of the HCC
low-risk gene expression and suppression of the HCC high-risk genes
(FIG. 7C). These results indicate that the CLDN1-specific mAbs
reverse HCC-risk gene expression induced by HBV infection and
suggest that the CLDN1-specific mAbs may exhibit chemopreventive
activity against HBV-induced HCC.
CLDN1-Specific mAbs Reverse the Patient-Derived Panetiology 32-Gene
HCC Risk Signature in Ethanol-Exposed Liver Cell-Based Model.
Next, to determine the potential of the anti-CLDN1 mAb for
prevention of HCC induced by alcohol consumption, the Applicants
assessed the ability of the antibody to reverse HCC-risk genes
modulated by a 10-day ethanol exposure of Huh7.5.1.sup.dif cells
(FIG. 8A). They thus investigated whether the 32-gene HCC-risk
signature (King et al., Gut, 2014, 64(8): 1296-1302) can be
reversed in Huh7.5.1.sup.dif cells following treatment of
ethanol-exposed cells with CLDN1-specific mAbs. Treatment with the
human CLDN1-specific mAb resulted in the induction of the HCC
low-risk gene expression and suppression of the HCC high-risk gene
expression in ethanol-exposed cells (FIG. 8B). These results
indicate that the CLDN1-specific mAbs reverse HCC-risk gene
expression induced by ethanol exposure and suggest that the
CLDN1-specific mAbs may exhibit chemopreventive activity against
HCC induced by alcohol consumption.
CLDN1-Specific mAb Reverses the Warburg-Like Metabolic Shift
Associated with Increased Cancer Risk and Cancer in the Liver
Cell-Based Model.
Mass spectrometry-based metabolomic profiling of HCV Jc1-infected
Huh7.5.1.sup.dif cells revealed alteration of steady-state
metabolite pools in hepatocytes, including pronounced effects on
lactate. Furthermore, metabolic labeling analysis demonstrated an
elevation of the lactate influx into HCV Jc1-infected
hepatocytes--a known Warburg-like metabolic shift associated with
malignant transformation and cancer (Cantor et al., Cancer
discovery, 2012, 2(10): 881-898). To assess whether the human
CLDN1-specific mAb is able to reverse this metabolic shift
associated with increased cancer risk and cancer, Huh7.5.1.sup.dif
cells were chronically infected with HCV and then treated with the
humanized CLDN1-specific mAb prior to metabolite analysis by mass
spectrometry (FIG. 9A). The metabolic profile of the CLDN1-specific
mAb-treated cells clusters with mock infected cells indicated that
the humanized CLDN1-specific Ab reverses the HCV-induced metabolic
shift in liver-derived cells (data not shown). Metabolic labeling
analysis demonstrated that the HCV-induced lactate flux is restored
to the level of the uninfected cells upon CLDN1-specific mAb
treatment (FIG. 9B). Furthermore, the expressions of several
metabolites belonging to the Krebs cycle were restored to their
levels of expression in uninfected cells following CLDN1-specific
mAb treatment of HCV-infected cells (FIG. 9C). Taken together,
these data indicate that the CLDN1-specific mAb reverses the
metabolic shift associated malignant transformation or cancer. This
suggests that the CLDN1-specific mAb may prevent malignant
transformation of hepatocytes as well as treat HCC.
CLDN1-Specific mAb Treatment Reverses Epithelial to Mesenchymal
(EMT) Transition Regulators.
EMT is an early step in metastasis, during which cancer cells lose
polarity and undergo rearrangement of cytoskeletal and cell
junction proteins (Lamouille et al., Nature Reviews Molecular Cell
Biology, 2014, 15(3): 178-196). It is induced by transcription
factors Snail, Slug and Zeb1 which have been associated with the
invasiveness of the tumor (Nie et al., Oncogene, 2015, doi:
10.1038/onc.2015.428). To further study the potential of
CLDN1-specific mAbs to prevent cancer development or treat HCC, the
Applicants assessed the ability of the humanized CLDN1-specific mAb
to modulate the expression of genes involved in EMT.
Huh7.5.1.sup.dif cells were chronically infected with HCV Jc1 and
then treated with the humanized CLDN1-specific mAb (FIG. 10A) prior
to assessment of the expression of genes induced during EMT. The
expression of SNAI1, SNAI2, and ZEB1 was down-regulated by the
humanized CLDN1-specific mAb (FIG. 10B). Furthermore, genes
belonging to the HCC 186-gene signature and dysregulated in the
course of EMT (Anastassiou et al., BMC cancer. 2011; 11:529; Medici
et al., Molecular Biology of the Cell, 2008, 19(11):4875-4887) were
reversed upon CLDN1-specific mAb treatment (FIG. 10C). These
results show that the CLDN1-specific mAb reverses the expression of
genes involved in EMT suggesting that the CLDN1-specific
mAb-mediated limitation of the progression of EMT could contribute
to prevent the progression of HCC development as well as treat
established HCC.
Conclusion
Collectively, the data presented here demonstrate that human, rat
and mouse CLDN1-specific mAbs reverse the expression of genes of
HCC risk signature which robustly predicts liver disease and HCC
development in patients with various etiologies of HCC. The
functional impact of the mAb for prevention and treatment of liver
disease progression and HCC development was demonstrated by showing
that mAb treatment resulted in the reduction of the expression of
genes involved in EMT progression, as well as a reversal of the
Warburg-like metabolic shift in liver cells associated with
malignant transformation and cancer. Since the CLDN1-specific mAbs
reverse the HCC-risk gene expression induced by the main causes of
HCC (HCV infection, HBV infection and ethanol), the mAbs are
suitable to prevent and/or treat HCC independent of its etiology
including viral, metabolic and other causes.
Example 3
Materials and Methods
5 week-old C3H/He male mice (Janvier Labs, Saint Berthevin, France)
received a single intraperitoneal injection of diethylnitrosamine
(DEN, Sigma Aldrich, Saint-Quentin Fallavier, France), a
well-established animal model for progressive liver disease and HCC
(Frey et al., Carcinogenesis, 2000, 21: 161-166). At week 18
post-injection, two mice were sacrificed to evaluate the induction
of liver disease (FIG. 11A). Between weeks 18 and 23, the remaining
mice received weekly intraperitoneal injections of PBS or mouse
anti-human CLDN1 mAb (20 mg/kg) for 5 weeks (FIG. 11A). One week
after the fifth injection of anti-CLDN1 mAb, i.e. at week 23
post-DEN injection, all mice were sacrificed and the livers were
harvested and a part was fixed in formalin for FFPE histological
analysis (hematoxilin/eosin staining) as well as trichrome
staining.
Results
To evaluate the effect of Claudin-1 specific mAb for prevention and
treatment of progressive liver disease and HCC in vivo, the
Applicants used the diethylnitrosamine (DEN) mouse model for liver
disease and HCC. DEN is a carcinogenic chemical, which has been
shown to robustly induce liver steatosis, fibrosis, cirrhosis and
HCC in animal models. DEN models have been successfully used for
proof-of-concept studies of drug treatment of liver disease
progression and of HCC chemopreventive drugs (Fuchs et al.,
Hepatology, 2014, 59: 1577-1590; Ip et al., Cancer Prev. Res.
(Phila), 2013, 6: 1304-1306; Haider et al., Mol. Cancer Ther.,
2013, 12(10): 1947-1957; Park et al., J. Cell Physiol., 2012,
227(3): 899-908).
In C3H/Ha mice, a single DEN administration resulted in
microvacuolar steatosis affecting around 10% of the liver as shown
by histopathology analyses performed at week 18 post DEN (FIG. 11B,
arrows). Liver steatosis was observed in a focal pattern and was
localized predominantly periportal (FIG. 11B, left panel).
Furthermore, DEN resulted in liver carcinogenesis as shown with
development of a liver tumor post DEN (FIG. 11E).
The effect of the CLDN1-specific antibody on liver disease and
carcinogenesis was then studied after animals had received antibody
treatment for five weeks. Whereas all control animals continued to
develop liver steatosis at week 23 post DEN administration, no
steatosis was observed in mice having received treatment with the
CLDN1-specific antibody (FIGS. 11C, D). Similarly, while a tumor
nodule was observed at the surface of the liver of a control mouse
(mouse #8841 in FIG. 11E), no tumor was detected in any mice
treated with the CLDN1-specific antibody (data not shown).
Collectively, these results demonstrate that the anti-CLDN1 mAb
reverses liver steatosis, improves liver disease and provides a HCC
chemopreventive effect in a state-of-the-art mouse model for
progressive liver disease and HCC.
Other Embodiments
Other embodiments of the invention will be apparent to those
skilled in the art from a consideration of the specification or
practice of the invention disclosed herein. It is intended that the
specification and examples be considered as exemplary only, with
the true scope of the invention being indicated by the following
claims.
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